Constructs having a sirp-alpha domain or variant thereof

ABSTRACT

The present disclosure features signal-regulatory protein α (SIRP-α) polypeptides and constructs that are useful, e.g., to target a cell (e.g., a cancer cell or a cell of the immune system), to increase phagocytosis of the target cell, to eliminate immune cells such as regulatory T-cells, to kill cancer cells, to treat a disease (e.g., cancer) in a subject, or any combinations thereof. The SIRP-α constructs include a high affinity SIRP-α D1 domain or variant thereof that binds CD47 with higher affinity than a wild-type SIRP-α. The SIRP-α polypeptides or constructs include a SIRP-α D1 variant fused to an Fc domain monomer, a human serum albumin (HSA), an albumin-binding peptide, or a polyethylene glycol (PEG) polymer. Compositions provided herein include (i) a polypeptide including a signal-regulatory protein α (SIRP-α) D1 variant and (ii) an antibody.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.16/153,404, filed Oct. 5, 2018, which is a continuation of U.S. patentapplication Ser. No. 15/230,186, filed Aug. 5, 2016 (U.S. Pat. No.10,259,859), which claims the benefit of U.S. Provisional ApplicationNo. 62/202,772, filed Aug. 7, 2015; U.S. Provisional Application No.62/202,775, filed Aug. 7, 2015; U.S. Provisional Application No.62/202,779, filed Aug. 7, 2015; U.S. Provisional Application No.62/276,801, filed Jan. 8, 2016; U.S. Provisional Application No.62/265,887 filed Dec. 10, 2015; U.S. Provisional Application No.62/276,796 filed Jan. 8, 2016; and U.S. Provisional Application No.62/346,414 filed Jun. 6, 2016 which applications are each incorporatedherein in their entireties by reference.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 757972000402SEQLIST.TXT,date recorded: Jan. 6, 2020, size: 427 KB)

SUMMARY OF THE INVENTION

Disclosed herein, in certain embodiments, are polypeptides comprising: asignal-regulatory protein α (SIRP-α) D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92. In some embodiments, the wild type SIRP-α D1 domain hasa sequence according to any one of SEQ ID NOs: 1-10. In someembodiments, the SIRP-α D1 domain comprises between one and nineadditional amino acid mutations relative to a wild-type SIRP-α D1 domainat a residue selected from the group consisting of: residue 6, residue27, residue 31, residue 47, residue 53, residue 54 residue 56, residue66, and residue 92. In some embodiments, the SIRP-α D1 variant comprisesthe amino acid sequence,EEELQXIlQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅X₆GX₇FPRVTTVSDXsTKRNNMDFSIRIGX₉ITPADAGTYYCX₁₀KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ IDNO: 49), wherein X₁ is V, L, or I; X₂ is A, I, V, or L; X₃ is I, F, S,or T; X₄ is E, V, or L; X₅ is K or R; X₆ is E or Q; X₇ is H, P, or R; X₈is L, T, S, or G; X₉ is A; and X₁₀ is V or I; and wherein the SIRP-α D1variant has at least two amino acid substitutions relative to awild-type SIRP-α D1 domain having a sequence according to SEQ ID NO: 1.In some embodiments, the SIRP-α D1 variant has an amino acid sequenceaccording to any one of SEQ ID NOs: 78-85. In some embodiments, theSIRP-α D1 variant comprises the amino acid sequence,EEELQXIlQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅X₆GX₇FPRVTTVSDX₈TKRNNMDFSIRIGX₉X₁₀X₁₁X₁₂ADAGTYYCX₁₃KFRKGSPDDVEFKSGAGTELSVR AKPS(SEQ ID NO: 218), wherein X₁ is V, L, or I; X₂ is A, V, L, or I; X₃ isI, S, T, or F; X₄ is E, L, or V; X₅ is K or R; X₆ is E or Q; X₇ is H, R,or P; X₈ is S, G, L, or T; X₉ is any amino acid; X₁₀ is any amino acid;X₁₁ is any amino acid; X₁₂ is any amino acid; and X₁₃ is V or I; andwherein the SIRP-α D1 variant has at least two amino acid substitutionsrelative to a wild-type SIRP-α D1 domain having a sequence according toSEQ ID NO: 1. In some embodiments, X₉ is A. In some embodiments, X₉ isN. In some embodiments, X₁₀ is I. In some embodiments, X₉ is N and X₁₀is P. In some embodiments, X₉ is N and X₁₁ is any amino acid other thanS, T, or C. In some embodiments, X₁₁ is T. In some embodiments, X₁₁ isan amino acid other than T. In some embodiments, X₁₂ is P. In someembodiments, X₉ is N and X₁₂ is any amino acid other than P. In someembodiments, the SIRP-α D1 variant comprises the amino acid sequence,EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅X₆GX₇FPRVTTVSDX₈TKRNNMDFSIRIGX₉ITXioADAGTYYCX₁₁KFRKGSPDDVEFKSGAGTELSVRAKP S (SEQID NO: 219), wherein X₁ is V, L, or I; X₂ is A, V, L, or I; X₃ is I, S,T, or F; X₄ is E, L, or V; X₅ is K or R; X₆ is E or Q; X₇ is H, R, or P;X₈ is S, G, L, or T; X₉ is N; X₁₀ is any amino acid other than P; andX₁₁ is V or I; and wherein the SIRP-α D1 variant has at least two aminoacid substitutions relative to a wild-type SIRP-α D1 domain having asequence according to SEQ ID NO: 1. In some embodiments, the SIRP-α D1variant comprises the amino acid sequence,EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRELIYNQX₄EGX₅FPRVTTVSDX₆TKRNNMDF SIRIGX₇ITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKP S (SEQ IDNO: 52), wherein X₁ is V, L, or I; X₂ is A, I, or L; X₃ is I, T, S, orF; X₄ is K or R; X₅ is H, P, or R; X₆ is L, T, or G; and X₇ is A; andwherein the SIRP-α D1 variant has at least two amino acid substitutionsrelative to a wild-type SIRP-α D1 domain having a sequence according toSEQ ID NO: 1. In some embodiments, X₁ is V or I, X₂ is A or I, X₃ is Ior F, X₄ is K or R, X₅ is H or P, X₆ is L or T, and X₇ is A. In someembodiments, the SIRP-α D1 variant has at least three amino acidsubstitutions relative to a wild-type SIRP-α D1 domain having a sequenceaccording to SEQ ID NO: 1. In some embodiments, the SIRP-α D1 varianthas at least four amino acid substitutions relative to a wild-typeSIRP-α D1 domain having a sequence according to SEQ ID NO: 1. In someembodiments, the SIRP-α D1 variant has at least five amino acidsubstitutions relative to a wild-type SIRP-α D1 domain having a sequenceaccording to SEQ ID NO: 1. In some embodiments, the SIRP-α D1 varianthas at least six amino acid substitutions relative to a wild-type SIRP-αD1 domain having a sequence according to SEQ ID NO: 1. In someembodiments, the SIRP-α D1 variant has at least seven amino acidsubstitutions relative to a wild-type SIRP-α D1 domain having a sequenceaccording to SEQ ID NO: 1. In some embodiments, X₁ is I. In someembodiments, X₂ is I. In some embodiments, X₃ is F. In some embodiments,X₄ is R. In some embodiments, X₅ is P. In some embodiments, X₆ is T. Insome embodiments, each of X₁, X₂, X₃, X₄, X₅, and X₆ is not a wild-typeamino acid. In some embodiments, the SIRP-α D1 variant has an amino acidsequence according to any one of SEQ ID NOs: 81-85. In some embodiments,the SIRP-α D1 variant comprises the amino acid sequence,EEELQX₁IQPDKSVSVAAGESAILHCTX₂TSLX₃PVGPIQWFRGAGPARELIYNQX₄EGX₅FPRVTTVSEX₆TKRENMDFSISISX₇ITPADAGTYYCVKFRKGSPDTEFKSGAGTEL SVRAKP S(SEQ ID NO: 212), wherein X₁ is V, L, or I; X₂ is V, I, or L; X₃ is I,T, S, or F; X₄ is K or R; X₅ is H, P, or R; X₆ is S, T, or G; and X₇ isA; and wherein the SIRP-α D1 variant has at least two amino acidsubstitutions relative to a wild-type SIRP-α D1 domain having thesequence of SEQ ID NO: 2. In some embodiments, the polypeptide binds tohuman CD47 with a K_(D) less than about 5×10⁻⁹ M. In some embodiments,the polypeptide further comprises an Fc domain monomer linked to theN-terminus or the C-terminus of the polypeptide, wherein the Fc domainmonomer is a human IgG1, IgG2, or IgG4 Fc region. In some embodiments,the Fc domain monomer comprises at least one mutation relative to awild-type human IgG1, IgG2, or IgG4 Fc region. In some embodiments, thepolypeptide has the amino acid sequence of any one of SEQ ID NO: 135,SEQ ID NO: 136, or SEQ ID NO: 137. In some embodiments, the Fc domainmonomer comprises (a)_one of the following amino acid substitutionsrelative to wild type human IgG1: T366W, T366S, L368A, Y407V, T366Y,T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L351K, P353S,S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T,L368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T,V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T,Y407H, Y407I, K409E, K409D, K409T, or K409I; or (b) (i) a N297A mutationrelative to a human IgG1 Fc region; (ii) a L234A, L235A, and G237Amutation relative to a human IgG1 Fc region; (iii) a L234A, L235A,G237A, and N297A mutation relative to a human IgG1 Fc region; (iv) aN297A mutation relative to a human IgG2 Fc region; (v) a A330S and P331S mutation relative to a human IgG2 Fc region; (vi) a A330S, P331 S, andN297A mutation relative to a human IgG2 Fc region; (vii) a S228P, E233P,F234V, L235A, and delG236 mutation relative to a human IgG4 Fc region;or (viii) a S228P, E233P, F234V, L235A, delG236, and N297A mutationrelative to a human IgG4 Fc region. In some embodiments, the Fc domainmonomer comprises (a) one of the following amino acid substitutionsrelative to wild type human IgG1: T366W, T366S, L368A, Y407V, T366Y,T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L351K, P353S,S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T,L368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T,V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T,Y407H, Y407I, K409E, K409D, K409T, or K409I; and (b) the Fc domainmonomer further comprises (i) a N297A mutation relative to a human IgG1Fc region; (ii) a L234A, L235A, and G237A mutation relative to a humanIgG1 Fc region; (iii) a L234A, L235A, G237A, and N297A mutation relativeto a human IgG1 Fc region; (iv) a N297A mutation relative to a humanIgG2 Fc region; (v) a A330S and P331S mutation relative to a human IgG2Fc region; (vi) a A330S, P331 S, and N297A mutation relative to a humanIgG2 Fc region; (vii) a S228P, E233P, F234V, L235A, and delG236 mutationrelative to a human IgG4 Fc region; or (viii) a S228P, E233P, F234V,L235A, delG236, and N297A mutation relative to a human IgG4 Fc region.In some embodiments, the polypeptide exhibits a reduction ofphagocytosis in a phagocytosis assay compared to a polypeptide with awild-type human IgG Fc region. In some embodiments, the Fc domainmonomer is linked to a second polypeptide comprising a second Fc domainmonomer to form an Fc domain dimer. In some embodiments, the second Fcdomain monomer is linked to an additional polypeptide. In someembodiments, the additional polypeptide comprises an antibody variabledomain. In some embodiments, the antibody variable domain targets anantigen expressed on a cell. In some embodiments, the cell is a cancercell. In some embodiments, the antibody variable domain targets a cellsurface protein involved in immune cell regulation. In some embodiments,the additional polypeptide comprises a therapeutic protein. In someembodiments, the therapeutic protein is a cytokine, an interleukin, anantigen, a steroid, an anti-inflammatory agent, or an immunomodulatoryagent. In some embodiments, the additional polypeptide comprises aSIRP-α D1 variant. In some embodiments, the polypeptide furthercomprises a human serum albumin (HSA) (SEQ ID NO: 12). In someembodiments, the HSA comprises a C34S or K573P amino acid substitutionrelative to SEQ ID NO: 12. In some embodiments, the polypeptide has anamino acid sequence according to any one of SEQ ID NOs: 152-159. In someembodiments, the polypeptide further comprises an albumin-bindingpeptide. In some embodiments, the albumin-binding peptide comprises theamino acid sequence DICLPRWGCLW (SEQ ID NO: 160). In some embodiments,the polypeptide further comprises a polyethylene glycol (PEG) polymer.In some embodiments, the PEG polymer is joined to a cysteinesubstitution in the polypeptide.

Disclosed herein, in certain embodiments, are polypeptides comprising: asignal-regulatory protein α (SIRP-α) D1 variant, wherein the SIRP-α D1variant comprises the amino acid sequence,EEX₁X₂QX₃IQPDKX₄VXsVAAGEX₆X₇X₈LX₉CTX₁₀TSLX₁₁PVGPIQWFRGAGPX₁₂RX₁₃LIYNQX₁₄X₁₅GX₁₆FPRVTTVSX₁₇X₁₈TX₁₉RX₂₀NMDFX₂₁IX₂IX₂₃X₂₄ITX₂₅ADAGTYYCX₂₆KX₂₇RKGSPDX₂₈X₂₉EX₃₀KSGAGTELSVRX₃₁KPS (SEQ ID NO: 47), wherein X₁ is E, or G; X₂is L, I, or V; X₃ is V, L, or I; X₄ is S, or F; X₅ is L, or S; X₆ is S,or T; X₇ is A, or V; X₈ is I, or T; X₉ is H, R, or L; X₁₀ is A, V, I, orL; X₁₁ is I, T, S, or F; X₁₂ is A, or G; X₁₃ is E, V, or L; X₁₄ is K, orR; X₁₅ is E, or Q; X₁₆ is H, P, or R; X₁₇ is D, or E; X₁₈ is S, L, T, orG; X₁₉ is K, or R; X₂₀ is E, or N; X₂₁ is S, or P; X₂₂ is S, or R; X₂₃is S, or G; X₂₄ is any amino acid; X₂₅ is any amino acid; X₂₆ is V, orI; X₂₇ is F, L, or V; X₂₈ is D or absent; X₂₉ is T, or V; X₃₀ is F, orV; and X₃₁ is A, or G; and wherein the SIRP-α D1 variant has at leasttwo amino acid substitutions relative to a wild-type SIRP-α D1 domainhaving a sequence according to any one of SEQ ID NOs: 1 to 10; and an Fcvariant comprising an Fc domain dimer having two Fc domain monomers,wherein each Fc domain monomer independently is (i) a human IgG1 Fcregion comprising a N297A mutation; (ii) a human IgG1 Fc regioncomprising L234A, L235A, and G237A mutations; (iii) a human IgG1 Fcregion comprising L234A, L235A, G237A, and N297A mutations; (iv) a humanIgG2 Fc region comprising a N297A mutation; (v) a human IgG2 Fc regioncomprising A330S and P331 S mutations; (vi) a human IgG2 Fc regioncomprising A330S, P331 S, and N297A mutations; (vii) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, and delG236 mutations; or(viii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A,delG236, and N297A mutations. In some embodiments, one of the Fc domainmonomers in the Fc domain dimer comprises a human IgG1 Fc regioncomprising L234A, L235A, G237A, and N297A mutations. In someembodiments, the polypeptide comprises an amino acid sequence accordingto any one of SEQ ID NOs: 98-104, 107-113, 116-122, or 135-137. In someembodiments, the Fc variant exhibits ablated or reduced binding to anFcγ receptor compared to a wild-type version of a human IgG Fc region.In some embodiments, the IgG1 or IgG2 Fc variant exhibits ablated orreduced binding to CD16a, CD32a, CD32b, CD32c, and CD64 Fcγ receptorscompared to a wild-type version of a human IgG1 or IgG2 Fc region. Insome embodiments, the IgG4 Fc variant exhibits ablated or reducedbinding to CD16a and CD32b Fcγ receptors compared to a wild-type versionof the human IgG4 Fc region. In some embodiments, the IgG1 or IgG2 Fcvariant exhibits ablated or reduced binding to C1q compared to awild-type version of a human IgG1 or IgG2 Fc fusion. In someembodiments, the Fc variant binds to an Fcγ receptor with a K_(D)greater than about 5×10⁻⁶ M.

Disclosed herein, in certain embodiments, are polypeptides comprising anFc variant, wherein the Fc variant comprises an Fc domain dimer havingtwo Fc domain monomers, wherein each Fc domain monomer independently isselected from (i) a human IgG1 Fc region consisting of mutations L234A,L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting ofmutations A330S, P331S and N297A; or (iii) a human IgG4 Fc regioncomprising mutations S228P, E233P, F234V, L235A, delG236, and N297A. Insome embodiments, at least one of the Fc domain monomers is a human IgG1Fc region consisting of mutations L234A, L235A, G237A, and N297A. Insome embodiments, at least one of the Fc domain monomers is a human IgG2Fc region consisting of mutations A330S, P331S and N297A. In someembodiments, the Fc variant exhibits ablated or reduced binding to anFcγ receptor compared to the wild-type version of the human IgG Fcregion. In some embodiments, the Fc variant exhibits ablated or reducedbinding to CD16a, CD32a, CD32b, CD32c, and CD64 Fcγ receptors comparedto the wild-type version of the human IgG Fc region. In someembodiments, the Fc variant exhibits ablated or reduced binding to C1qcompared to the wild-type version of the human IgG Fc fusion. In someembodiments, at least one of the Fc domain monomers is a human IgG4 Fcregion comprising mutations S228P, E233P, F234V, L235A, delG236, andN297A. In some embodiments, the Fc variant exhibits ablated or reducedbinding to a Fcγ receptor compared to the wild-type human IgG4 Fcregion. In some embodiments, the Fc variant exhibits ablated or reducedbinding to CD16a and CD32b Fcγ receptors compared to the wild-typeversion of its human IgG4 Fc region. In some embodiments, the Fc variantbinds to an Fcγ receptor with a K_(D) greater than about 5×10⁻⁶ M. Insome embodiments, the polypeptide further comprises a CD47 bindingpolypeptide. In some embodiments, the Fc variant exhibits ablated orreduced binding to an Fcγ receptor compared to a wild-type version of ahuman IgG Fc region. In some embodiments, the CD47 binding polypeptidedoes not cause acute anemia in rodents and non-human primates. In someembodiments, the CD47 binding polypeptide does not cause acute anemia inhumans. In some embodiments, the CD47 binding polypeptide is asignal-regulatory protein α (SIRP-α) polypeptide or a fragment thereof.In some embodiments, the SIRP-α polypeptide comprises a SIRP-α D1variant comprising the amino acid sequence,EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅EGX₆FPRVTTVSDX₇TKRNNMDFSIRIGX₈ITPADAGTYYCX₉KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ IDNO: 51), wherein X₁ is V or I; X₂ is A or I; X₃ is I or F; X₄ is E or V;X₅ is K or R; X₆ is H or P; X₇ is L or T; X₈ is any amino acid otherthan N; and X₉ is V or I. In some embodiments, the SIRP-α polypeptidecomprises a SIRP-α D1 variant wherein X₁ is V or I; X₂ is A or I; X₃ isI or F; X₄ is E; X₅ is K or R; X₆ is H or P; X₇ is L or T; X₈ is not N;and X₉ is V.

Disclosed herein, in certain embodiments, are polypeptides comprising: asignal-regulatory protein α (SIRP-α) D1 variant, wherein the SIRP-α D1variant is a non-naturally occurring high affinity SIRP-α D1 domain,wherein the SIRP-α D1 variant binds to human CD47 with an affinity thatis at least 10-fold greater than the affinity of a naturally occurringD1 domain; and an Fc domain monomer, wherein the Fc domain monomer islinked to a second polypeptide comprising a second Fc domain monomer toform an Fc domain, wherein the Fc domain has ablated or reduced effectorfunction. In some embodiments, the non-naturally occurring high affinitySIRP-α D1 domain comprises an amino acid mutation at residue 80.

Disclosed herein, in certain embodiments, are polypeptides comprising asignal-regulatory protein α (SIRP-α) D1 variant, wherein the SIRP-α D1variant binds CD47 from a first species with a K_(D) less than 250 nM;and wherein the SIRP-α D1 variant binds CD47 from a second species witha K_(D) less than 250 nM; and the K_(D) for CD47 from the first speciesand the K_(D) for CD47 from the second species are within 100 fold ofeach other; wherein the first species and the second species areselected from the group consisting of: human, rodent, and non-humanprimate. In some embodiments, the SIRP-α D1 variant binds CD47 from atleast 3 different species. In some embodiments, the non-human primate iscynomolgus monkey.

Disclosed herein, in certain embodiments, are polypeptides comprising:(a) a signal-regulatory protein α (SIRP-α) D1 domain that binds humanCD47 with a K_(D) less than 250 nM; and (b) an Fc domain monomer linkedto the N-terminus or the C-terminus of the SIRP-α D1 domain, wherein thepolypeptide does not cause acute anemia in rodents and non-humanprimates. In some embodiments, the polypeptide is a non-naturallyoccurring variant of a human SIRP-α. In some embodiments, administrationof the polypeptide in vivo results in hemoglobin reduction by less than50% during the first week after administration. In some embodiments,administration of the polypeptide in humans results in hemoglobinreduction by less than 50% during the first week after administration.In some embodiments, the polypeptide further comprises at least one Fcvariant, wherein the Fc variant is selected from (i) a human IgG1 Fcregion consisting of mutations L234A, L235A, G237A, and N297A; (ii) ahuman IgG2 Fc region consisting of mutations A330S, P331 S and N297A; or(iii) a human IgG4 Fc region comprising mutations S228P, E233P, F234V,L235A, delG236, and N297A. In some embodiments, the Fc variant is ahuman IgG1 Fc region consisting of mutations L234A, L235A, G237A, andN297A. In some embodiments, the Fc variant is a human IgG2 Fc regionconsisting of mutations A330S, P331S and N297A. In some embodiments, theFc variant is a human IgG4 Fc region comprising mutations S228P, E233P,F234V, L235A, delG236, and N297A.

Disclosed herein, in certain embodiments, are methods of treating anindividual having a disease or disorder, the method comprisingadministering to the subject a polypeptide disclosed herein. In someembodiments, the polypeptide comprises a signal-regulatory protein α(SIRP-α) D1 variant comprising a SIRP-α D1 domain, or a fragmentthereof, having an amino acid mutation at residue 80 relative to awild-type SIRP-α D1 domain; and at least one additional amino acidmutation relative to a wild-type SIRP-α D1 domain at a residue selectedfrom the group consisting of: residue 6, residue 27, residue 31, residue47, residue 53, residue 54, residue 56, residue 66, and residue 92. Insome embodiments, the polypeptide comprises a signal-regulatory proteinα (SIRP-α) D1 variant, wherein the SIRP-α D1 variant comprises the aminoacid sequence,EEX₁X₂QX₃IQPDKX₄VX₅VAAGEX₆X₇X₈X₉CTX₁₀TSLX₁₁PVGPIQWFRGAGPX₁₂RX₁₃LIYNQX₁₄X₁₅GX₁₆FPRVTTVSX₁₇X₁₈TX₁₉RX₂₀NMDFX₂₁IX₂₂IX₂₃X₂₄ITX₂₅ADAGTYYCX₂₆KX₂₇RKGSPDX₂₈X₂₉EX₃₀KSGAGTELSVRX₃₁KPS (SEQ ID NO: 47), wherein X₁ is E, or G; X₂is L, I, or V; X₃ is V, L, or I; X₄ is S, or F; X₅ is L, or S; X₆ is S,or T; X₇ is A, or V; X₈ is I, or T; X₉ is H, R, or L; X₁₀ is A, V, I, orL; X₁₁ is I, T, S, or F; X₁₂ is A, or G; X₁₃ is E, V, or L; X₁₄ is K, orR; X₁₅ is E, or Q; X₁₆ is H, P, or R; X₁₇ is D, or E; X₁₈ is S, L, T, orG; X₁₉ is K, or R; X₂₀ is E, or N; X₂₁ is S, or P; X₂₂ is S, or R; X₂₃is S, or G; X₂₄ is any amino acid; X₂₅ is any amino acid; X₂₆ is V, orI; X₂₇ is F, L, or V; X₂₈ is D or absent; X₂₉ is T, or V; X₃₀ is F, orV; and X₃₁ is A, or G; and wherein the SIRP-α D1 variant has at leasttwo amino acid substitutions relative to a wild-type SIRP-α D1 domainhaving a sequence according to any one of SEQ ID NOs: 1 to 10; and an Fcvariant comprising an Fc domain dimer having two Fc domain monomers,wherein each Fc domain monomer independently is (i) a human IgG1 Fcregion comprising a N297A mutation; (ii) a human IgG1 Fc regioncomprising L234A, L235A, and G237A mutations; (iii) a human IgG1 Fcregion comprising L234A, L235A, G237A, and N297A mutations; (iv) a humanIgG2 Fc region comprising a N297A mutation; (v) a human IgG2 Fc regioncomprising A330S and P331S mutations; (vi) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations; (vii) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, and delG236 mutations; or(viii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A,delG236, and N297A mutations. In some embodiments, the polypeptidecomprises an Fc variant, wherein the Fc variant comprises an Fc domaindimer having two Fc domain monomers, wherein each Fc domain monomerindependently is selected from (i) a human IgG1 Fc region consisting ofmutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc regionconsisting of mutations A330S, P331S and N297A; or (iii) a human IgG4 Fcregion comprising mutations S228P, E233P, F234V, L235A, delG236, andN297A. In some embodiments, the polypeptide comprises asignal-regulatory protein α (SIRP-α) D1 variant, wherein the SIRP-α D1variant is a non-naturally occurring high affinity SIRP-α D1 domain,wherein the SIRP-α D1 variant binds to human CD47 with an affinity thatis at least 10-fold greater than the affinity of a naturally occurringD1 domain; and an Fc domain monomer, wherein the Fc domain monomer islinked to a second polypeptide comprising a second Fc domain monomer toform an Fc domain, wherein the Fc domain has ablated or reduced effectorfunction. In some embodiments, the non-naturally occurring high affinitySIRP-α D1 domain comprises an amino acid mutation at residue 80. In someembodiments, the polypeptide comprises a signal-regulatory protein α(SIRP-α) D1 variant, wherein the SIRP-α D1 variant binds CD47 from afirst species with a K_(D) less than 250 nM; and wherein the SIRP-α D1variant binds CD47 from a second species with a K_(D) less than 250 nM;and the K_(D) for CD47 from the first species and the K_(D) for CD47from the second species are within 100 fold of each other; wherein thefirst species and the second species are selected from the groupconsisting of: human, rodent, and non-human primate. In someembodiments, the polypeptide comprises (a) a signal-regulatory protein α(SIRP-α) D1 domain that binds human CD47 with a K_(D) less than 250 nM;and (b) an Fc domain monomer linked to the N-terminus or the C-terminusof the SIRP-α D1 domain, wherein the polypeptide does not cause acuteanemia in rodents and non-human primates. In some embodiments, thedisease or disorder is a cancer, an autoimmune disease, or aninflammatory disease. In some embodiments, the disease or disorder is acancer, and the cancer is selected from solid tumor cancer,hematological cancer, acute myeloid leukemia, chronic lymphocyticleukemia, chronic myeloid leukemia, acute lymphoblastic leukemia,non-Hodgkin lymphoma, Hodgkin lymphoma, multiple myeloma, bladdercancer, pancreatic cancer, cervical cancer, endometrial cancer, lungcancer, bronchus cancer, liver cancer, ovarian cancer, colon and rectalcancer, stomach cancer, gastric cancer, gallbladder cancer,gastrointestinal stromal tumor cancer, thyroid cancer, head and neckcancer, oropharyngeal cancer, esophageal cancer, melanoma, non-melanomaskin cancer, Merkel cell carcinoma, virally induced cancer,neuroblastoma, breast cancer, prostate cancer, renal cancer, renal cellcancer, renal pelvis cancer, leukemia, lymphoma, sarcoma, glioma, braintumor, and carcinoma. In some embodiments, the disease or disorder is anautoimmune disease or an inflammatory disease, and the autoimmunedisease or the inflammatory disease is selected from multiple sclerosis,rheumatoid arthritis, a spondyloarthropathy, systemic lupuserythematosus, an antibody-mediated inflammatory or autoimmune disease,graft versus host disease, sepsis, diabetes, psoriasis, atherosclerosis,Sjogren's syndrome, progressive systemic sclerosis, scleroderma, acutecoronary syndrome, ischemic reperfusion, Crohn's Disease, endometriosis,glomerulonephritis, myasthenia gravis, idiopathic pulmonary fibrosis,asthma, acute respiratory distress syndrome (ARDS), vasculitis, andinflammatory autoimmune myositis. In some embodiments, the SIRP-α D1variant has a sequence according to any one of SEQ ID NOs: 78-85,98-104, 107-113, 116-122, 135-137, or 152-159. In some embodiments, themethod further comprises administering at least one additional agent. Insome embodiments, the at least one additional agent is an antibody,tumor associated antigen, or a non-antibody therapeutic. In someembodiments, at least two additional agents are administered. In someembodiments, the at least two additional agents comprise two antibodies.In some embodiments, the at least two additional agents comprise anantibody and a tumor associated antigen. In some embodiments, the atleast one additional agent is an antibody. In some embodiments, theantibody is a human IgG1 isotype antibody. In some embodiments, theantibody is a human IgG2 isotype antibody. In some embodiments, theantibody is a human IgG4 isotype antibody. In some embodiments, theantibody is selected from an anti-HER2 antibody, anti-CD20 antibody,anti-CD19 antibody, anti-CS1 antibody, anti-CD38 antibody, anti-EGFRantibody, anti-PD1 antibody, anti-OX40 antibody, anti-PD-1 antibody,anti-PD-L1 antibody, anti-CD274 antibody, anti-CTLA-4 antibody,anti-CD137 antibody, anti-4-1BB antibody, anti-B7-H3 antibody, anti-FZD7antibody, anti-CD27 antibody, anti-CCR4 antibody, anti-CD38 antibody,anti-CSF1R antibody, anti-CSF antibody, anti-CD30 antibody, anti-BAFFantibody, anti-VEGF antibody, or anti-VEGFR2 antibody. In someembodiments, the antibody is selected from an anti-HER2 antibody,anti-CD20 antibody, anti-CD19 antibody, anti-CS1 antibody, anti-CD38antibody, anti-PD-1 antibody, anti-RANKL antibody, or anti-PD-L1antibody. In some embodiments, the at least one additional agent is atleast one antibody and the antibody is selected from cetuximab,necitumumab, pembrolizumab, nivolumab, pidilizumab, MEDI0680, MED16469,atezolizumab, avelumab, durvalumab, MEDI6383, RG7888, ipilimumab,tremelimumab, urelumab, PF-05082566, enoblituzumab, vantictumab,varlilumab, mogamalizumab, SAR650984, daratumumab, trastuzumab,trastuzumab emtansine, pertuzumab, elotuzumab, rituximab, ofatumumab,obinutuzumab, RG7155, FPA008, panitumumab, brentuximab vedotin,MSB0010718C, belimumab, bevacizumab, denosumab, panitumumab,ramucirumab, necitumumab, nivolumab, pembrolizumab, avelumab,atezolizumab, durvalumab, MEDI0680, pidilizumab, or BMS-93659. In someembodiments, the antibody is trastuzumab. In some embodiments, theSIRP-α D1 variant has a sequence according to any one of SEQ ID NOs:78-85, 98-104, 107-113, 116-122, 135-137, or 152-159. In someembodiments, the antibody is rituximab. In some embodiments, the SIRP-αD1 variant has a sequence according to any one of SEQ ID NOs: 78-85,98-104, 107-113, 116-122, 135-137, or 152-159. In some embodiments, theantibody is cetuximab. In some embodiments, the SIRP-α D1 variant has asequence according to any one of SEQ ID NOs: 78-85, 98-104, 107-113,116-122, 135-137, or 152-159. In some embodiments, the antibody isdaratumumab. In some embodiments, the SIRP-α D1 variant has a sequenceaccording to any one of SEQ ID NOs: 78-85, 98-104, 107-113, 116-122,135-137, or 152-159. In some embodiments, the antibody is belimumab. Insome embodiments, the SIRP-α D1 variant has a sequence according to anyone of SEQ ID NOs: 78-85, 98-104, 107-113, 116-122, 135-137, or 152-159.In some embodiments, n the antibody is bevacizumab. In some embodiments,the SIRP-α D1 variant has a sequence according to any one of SEQ ID NOs:78-85, 98-104, 107-113, 116-122, 135-137, or 152-159. In someembodiments, the antibody is denosumab. In some embodiments, the SIRP-αD1 variant has a sequence according to any one of SEQ ID NOs: 78-85,98-104, 107-113, 116-122, 135-137, or 152-159. In some embodiments, theantibody is pantimumab. In some embodiments, the SIRP-α D1 variant has asequence according to any one of SEQ ID NOs: 78-85, 98-104, 107-113,116-122, 135-137, or 152-159. In some embodiments, the antibody isramucirumab. In some embodiments, the SIRP-α D1 variant has a sequenceaccording to any one of SEQ ID NOs: 78-85, 98-104, 107-113, 116-122,135-137, or 152-159. In some embodiments, the antibody is necitumumab.In some embodiments, the SIRP-α D1 variant has a sequence according toany one of SEQ ID NOs: 78-85, 98-104, 107-113, 116-122, 135-137, or152-159. In some embodiments, the antibody is nivolumab. In someembodiments, the SIRP-α D1 variant has a sequence according to any oneof SEQ ID NOs: 78-85, 98-104, 107-113, 116-122, 135-137, or 152-159. Insome embodiments, the antibody is pembrolizumab. In some embodiments,the SIRP-α D1 variant has a sequence according to any one of SEQ ID NOs:78-85, 98-104, 107-113, 116-122, 135-137, or 152-159. In someembodiments, the antibody is avelumab. In some embodiments, the SIRP-αD1 variant has a sequence according to any one of SEQ ID NOs: 78-85,98-104, 107-113, 116-122, 135-137, or 152-159. In some embodiments, theantibody is atezolizumab. In some embodiments, the SIRP-α D1 variant hasa sequence according to any one of SEQ ID NOs: 78-85, 98-104, 107-113,116-122, 135-137, or 152-159. In some embodiments, the antibody isdurvalumab. In some embodiments, the SIRP-α D1 variant has a sequenceaccording to any one of SEQ ID NOs: 78-85, 98-104, 107-113, 116-122,135-137, or 152-159. In some embodiments, the antibody is MEDI0680. Insome embodiments, the SIRP-α D1 variant has a sequence according to anyone of SEQ ID NOs: 78-85, 98-104, 107-113, 116-122, 135-137, or 152-159.In some embodiments, the antibody is pidilizumab. In some embodiments,the SIRP-α D1 variant has a sequence according to any one of SEQ ID NOs:78-85, 98-104, 107-113, 116-122, 135-137, or 152-159. In someembodiments, the antibody is BMS-93659. In some embodiments, the SIRP-αD1 variant has a sequence according to any one of SEQ ID NOs: 78-85,98-104, 107-113, 116-122, 135-137, or 152-159. In some embodiments, theat least one additional agent is a tumor associated antigen and thetumor associated antigen elicits an immune response. In someembodiments, the at least one additional agent is an antibody and theantibody targets a HLA/peptide or MHC/peptide complex. In someembodiments, the antibody targets a HLA/peptide or MHC/peptide complexcomprising NY-ESO-1/LAGE1, SSX-2, MAGE family (MAGE-A3), gp100/pmel17,Melan-A/MART-1, gp75/TRP1, tyrosinase, TRP2, CEA, PSA, TAG-72, Immaturelaminin receptor, MOK/RAGE-1, WT-1, Her2/neu, EphA3, SAP-1, BING-4,Ep-CAM, MUC1, PRAME, survivin, Mesothelin, BRCA1/2 (mutated), CDK4,CML66, MART-2, p53 (mutated), Ras (mutated), β-catenin (mutated),TGF-βRII (mutated), HPV E6, or E7. In some embodiments, the antibody isESK1, RL1B, Pr20, or 3.2G1.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is an illustration of a SIRP-α construct including a SIRP-α D1domain or variant thereof joined to a first Fc domain monomer, whichforms an Fc domain with a second Fc domain monomer;

FIG. 2 is an illustration of a SIRP-α construct including a SIRP-α D1domain or variant thereof joined to a first Fc domain monomer and anantibody variable domain joined to a second Fc domain monomer, whereinthe first Fc domain monomer and the second Fc domain monomer combine toform an Fc domain;

FIG. 3 is an illustration of a SIRP-α construct including a SIRP-α D1domain or variant thereof joined to a first Fc domain monomer and atherapeutic protein joined to a second Fc domain monomer, wherein thefirst Fc domain monomer and the second Fc domain monomer combine to forman Fc domain;

FIG. 4A is an illustration of a SIRP-α construct including a SIRP-α D1domain or variant thereof joined to a first Fc domain monomer having aknob mutation, which forms an Fc domain with a second Fc domain monomerhaving a hole mutation; FIG. 4B is an illustration of a SIRP-α constructincluding a SIRP-α D1 domain or variant thereof joined to a first Fcdomain monomer having a hole mutation, which forms an Fc domain with asecond Fc domain monomer having a knob mutation;

FIG. 5A is an illustration of a SIRP-α construct including a SIRP-α D1domain or variant thereof joined to an Fc domain monomer; FIG. 5B is anillustration of a SIRP-α construct which is a homodimer of the constructillustrated in FIG. 5A;

FIG. 6 exemplifies SPR binding data for monofunctional and bifunctionalSIRP-α constructs including a SIRP-α D1 domain;

FIG. 7 exemplifies phagocytosis of DLD-1-GFP-Luciferase tumor cells byhuman monocyte-derived macrophages in the presence of varyingconcentrations of SIRP-α polypeptide constructs;

FIG. 8 exemplifies phagocytosis of DLD-1-GFP-Luciferase tumor cells byhuman monocyte-derived macrophages in the presence of varyingconcentrations of SIRP-α polypeptide constructs;

FIG. 9 exemplifies phagocytosis of DLD-1-GFP-Luciferase tumor cells byhuman monocyte-derived macrophages in the presence of varyingconcentrations of SIRP-α polypeptide constructs;

FIG. 10 exemplifies half-life stability of SIRP-α polypeptides over adefined time period;

FIG. 11 exemplifies hemagglutination assay data for SIRP-α polypeptideconstructs;

FIG. 12 exemplifies survival curves of mice syngeneic tumor modelstreated with SIRP-α polypeptide constructs and anti-mPD-L1;

FIG. 13 exemplifies a tumor volume analysis of mice syngeneic tumormodels treated with SIRP-α polypeptide constructs in combination withanti-mPD-L1;

FIG. 14 exemplifies binding of various concentrations of C1q complementto SIRP-α-Fc fusions;

FIG. 15 exemplifies phagocytosis of MM1R cells by human monocyte-derivedmacrophages in the presence of varying concentrations of SIRP-αpolypeptide constructs;

FIG. 16 exemplifies phagocytosis of MM1R cells by human monocyte-derivedmacrophages in the presence of varying concentrations of SIRP-αpolypeptide constructs;

FIG. 17 exemplifies phagocytosis of N87 cells by human monocyte-derivedmacrophages in the presence of varying concentrations of SIRP-αpolypeptide constructs;

FIG. 18 exemplifies molecular weight analysis of a SIRP-α D1 varianthaving a P83V mutation;

FIG. 19A exemplifies tumor growth of human GFP-Luc-Raji lymphoma cellsin a NOD scid gamma (NSG) mouse model of cancer treated with variousSIRP-α constructs with or without rituximab; FIG. 19B exemplifies ascatter plot of tumor volume of the tumors described in FIG. 19A; FIG.19C exemplifies hemoglobin values of the treated mice described in FIG.19A; and

FIG. 20 exemplifies red blood cell counts taken from mice treated witheither a SIRP-α wildtype IgG1 Fc construct or a SIRP-α IgG1 Fc variantconstruct.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, up to 10%, up to 5%, or up to 1% of a given value.Alternatively, particularly with respect to biological systems orprocesses, the term can mean within an order of magnitude, preferablywithin 5-fold, and more preferably within 2-fold, of a value. Whereparticular values are described in the application and claims, unlessotherwise stated the term “about” meaning within an acceptable errorrange for the particular value should be assumed.

The terminology used herein is for the purpose of describing particularcases only and is not intended to be limiting. As used herein, thesingular forms “a”, “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.Furthermore, to the extent that the terms “including”, “includes”,“having”, “has”, “with”, or variants thereof are used in either thedetailed description or the claims, such terms are intended to beinclusive in a manner similar to the term “comprising.”

As used herein, the term “antibody” refers to intact antibodies;antibody fragments, provided that they exhibit the desired biologicalactivity (e.g. epitope binding); monoclonal antibodies; polyclonalantibodies; monospecific antibodies; multi-specific antibodies (e.g.,bispecific antibodies); and antibody-like proteins.

As used herein, the term “antibody variable domain” refers to theportions of the light and heavy chains of an antibody that include aminoacid sequences of complementary determining regions (CDRs, e.g., CDR L1,CDR L2, CDR L3, CDR H1, CDR H2, and CDR H3) and framework regions (FRs).

As used herein, the term “linker” refers to a linkage between twoelements, e.g., protein domains. In some embodiments, a linker can be acovalent bond or a spacer. The term “spacer” refers to a moiety (e.g., apolyethylene glycol (PEG) polymer) or an amino acid sequence (e.g., a1-200 amino acid sequence) occurring between two polypeptides orpolypeptide domains to provide space or flexibility (or both space andflexibility) between the two polypeptides or polypeptide domains. Insome embodiments, an amino acid spacer is part of the primary sequenceof a polypeptide (e.g., joined to the spaced polypeptides or polypeptidedomains via the polypeptide backbone).

As used herein, the term “therapeutically effective amount” refers to anamount of a polypeptide or a pharmaceutical composition containing apolypeptide described herein, e.g., a polypeptide having a SIRP-α D1domain or variant thereof, that is sufficient and effective in achievinga desired therapeutic effect in treating a patient having a disease,such as a cancer, e.g., solid tumor or hematological cancer. In someembodiments, a therapeutically effective amount of polypeptide willavoid adverse side effects.

As used herein, the term “pharmaceutical composition” refers to amedicinal or pharmaceutical formulation that includes an activeingredient as well as excipients or diluents (or both excipients anddiluents) and enables the active ingredient to be administered bysuitable methods of administration. In some embodiments, thepharmaceutical compositions disclosed herein include pharmaceuticallyacceptable components that are compatible with the polypeptide. In someembodiments, the pharmaceutical composition is in tablet or capsule formfor oral administration or in aqueous form for intravenous orsubcutaneous administration, for example by injection.

As used herein, the terms “subject,” “individual,” and “patient” areused interchangeably to refer to a vertebrate, for example, a mammal.Mammals include, but are not limited to, murines, simians, humans, farmanimals, sport animals, and pets. Tissues, cells, and their progeny of abiological entity obtained in vivo or cultured in vitro are alsoencompassed. None of the terms entail supervision of a medicalprofessional.

As used herein, the term “affinity” or “binding affinity” refers to thestrength of the binding interaction between two molecules. Generally,binding affinity refers to the strength of the sum total of non-covalentinteractions between a molecule and its binding partner, such as a highaffinity SIRP-α D1 variant and CD47. Unless indicated otherwise, bindingaffinity refers to intrinsic binding affinity, which reflects a 1:1interaction between members of a binding pair. The binding affinitybetween two molecules is commonly described by the dissociation constant(K_(D)) or the association constant (K_(A)). Two molecules that have lowbinding affinity for each other generally bind slowly, tend todissociate easily, and exhibit a large K_(D). Two molecules that havehigh affinity for each other generally bind readily, tend to remainbound longer, and exhibit a small K_(D). In some embodiments, the K_(D)of two interacting molecules is determined using known methods andtechniques, e.g., surface plasmon resonance (SPR). K_(D) can becalculated as the ratio of k_(off)/k_(on).

As used herein, the term “K_(D) less than” refers to a numericallysmaller K_(D) value and an increasing binding affinity relative to therecited K_(D) value. As used herein, the term “K_(D) greater than”refers to a numerically larger K_(D) value and a decreasing bindingaffinity relative to the recited K_(D) value.

As used herein, the term “acute anemia” refers to a decrease of redblood cell mass or hemoglobin of 30% during the first five days afteradministration of a compound or treatment.

I. Signal-Regulatory Protein α (SIRP-α) D1 Domain and Variants Thereof

Disclosed herein, in some embodiments, are polypeptides comprising asignal-regulatory protein α (SIRP-α) D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92.

Also disclosed herein, in some embodiments, are polypeptides comprisingan Fc variant, wherein the Fc variant comprises an Fc domain dimerhaving two Fc domain monomers, wherein each Fc domain monomerindependently is selected from (i) a human IgG1 Fc region consisting ofmutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc regionconsisting of mutations A330S, P331S and N297A; or (iii) a human IgG4 Fcregion comprising mutations S228P, E233P, F234V, L235A, delG236, andN297A.

Signal-regulatory protein α (“SIRP-α” or “SIRP-alpha”) is atransmembrane glycoprotein belonging to the Ig superfamily that iswidely expressed on the membrane of myeloid cells. SIRP-α interacts withCD47, a protein broadly expressed on many cell types in the body. Theinteraction of SIRP-α with CD47 prevents engulfment of “self” cells,which can otherwise be recognized by the immune system. It has beenobserved that high CD47 expression on tumor cells can act, in acutemyeloid leukemia and several solid tumor cancers, as a negativeprognostic factor for survival.

Native SIRP-α comprises 3 highly homologous immunoglobulin (Ig)-likeextracellular domains—D1, D2, and D3. The SIRP-α D1 domain (“D1 domain”)refers to the membrane distal, extracellular domain of SIRP-α andmediates binding of SIRP-α to CD47. As used herein, the term “SIRP-αpolypeptide” refers to any SIRP-α polypeptide or fragment thereof thatis capable of binding to CD47. There are at least ten variants ofwild-type human SIRP-α. Table 1 shows the amino acid sequences of the D1domains of the ten naturally occurring wild-type human SIRP-α D1 domainvariants (SEQ ID NOs: 1-10). In some embodiments, a SIRP-α polypeptidecomprises a SIRP-α D1 domain. In some embodiments, a SIRP-α polypeptidecomprises a wild-type D1 domain, such as those provided in SEQ ID NOs:1-10. In some embodiments, a SIRP-α polypeptide includes a D2 or D3domain (or both a D2 and a D3 domain) (Table 3) of a wild-type humanSIRP-α.

TABLE 1 Sequences of Wild-Type SIRP-α D1 Domains SEQ ID NO: DescriptionAmino Acid Sequence  1 Wild-type D1 EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIdomain variant 1 QWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGA GTELSVRAKPS  2 Wild-type D1EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPI domain variant 2QWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAG TELSVRAKPS  3 Wild-type D1EEELQVIQPDKSVSVAAGESAILLCTVTSLIPVGPI domain variant 3QWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAG TELSVRAKPS  4 Wild-type D1EEGLQVIQPDKSVSVAAGESAILHCTATSLIPVGPI domain variant 4QWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGA GTELSVRAKPS  5 Wild-type D1EEELQVIQPDKFVLVAAGETATLRCTATSLIPVGPI domain variant 5QWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGA GTELSVRAKPS  6 Wild-type D1EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPI domain variant 6QWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFPIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGA GTELSVRAKPS  7 Wild-type D1EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPI domain variant 7QWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAG TELSVRGKPS  8 Wild-type D1EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPI domain variant 8QWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAG TELSVRAKPS  9 Wild-type D1EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPI domain variant 9QWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRISNITPADAGTYYCVKFRKGSPDDVEFKSGA GTELSVRAKPS 10 Wild-type D1EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPI domain variant 10QWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAG TELSVRAKPS 11 Wild-type pan-D1EEX₁LQVIQPDKX₂VX₃VAAGEX₄AX₅LX₆CTX₇TSLI domainPVGPIQWFRGAGPX₈RELIYNQKEGHFPRVTTVSX₉X₁₀TKRX₁₁NMDFX₁₂IX₁₃IX₁₄NITPADAGTYYCVKFRKGSX₁₅X₁₆DX₁₇EFKSGAGTELSVRX₁₈KPS Amino acid X₁ is E or G; X₂ is S or F;substitutions X₃ is L or S; X₄ is T or S; relative toX₅ is T or 1; X₆ is R, H, or L; SEQ ID NO: 11X₇ is A or V; X₈ is G or A; X₉ is D or E; X₁₀ is L or S;X₁₁ is N or E or D; X₁₂ is S or P; X₁₃ is R or S; X₁₄ is G or S;X₁₅ is P or absent; X₁₆ is D or P; X₁₇ is V or T; and X₁₈ is A or G

As used herein, the terms “high affinity SIRP-α D1 variant,” “highaffinity SIRP-α variant,” or “SIRP-α D1 variant” refers to a polypeptidecomprising a SIRP-α D1 domain or a CD47-binding portion of a SIRP-αpolypeptide that has a higher affinity to CD47 than wild-type SIRP-α. Ahigh affinity SIRP-α D1 variant comprises at least one amino acidsubstitution, deletion, or insertion (or a combination thereof) relativeto a wild-type SIRP-α.

In some embodiments, high affinity SIRP-α D1 variants disclosed hereincomprise a SIRP-α D1 domain or variant thereof. In some embodiments, ahigh affinity SIRP-α D1 variant comprises one or more amino acidsubstitutions, insertions, additions, or deletions relative to awild-type D1 domain shown in SEQ ID NOs: 1-10. Table 2 lists exemplaryamino acid substitutions in each SIRP-α D1 domain variant (SEQ ID NOs:13-22). In some embodiments, the SIRP-α D1 domain polypeptide or highaffinity SIRP-α D1 variant comprises a fragment of the D1 domain. Insome embodiments, the SIRP-α polypeptide fragment or high affinitySIRP-α variant fragment comprises an amino acid sequence of less than 10amino acids in length, about 10 amino acids in length, about 20 aminoacids in length, about 30 amino acids in length, about 40 amino acids inlength, about 50 amino acids in length, about 60 amino acids in length,about 70 amino acids in length, about 80 amino acids in length, about 90amino acids in length, about 100 amino acids in length, or more thanabout 100 amino acids in length. In some embodiments, the SIRP-α D1domain fragments retain the ability to bind to CD47.

In some embodiments, a polypeptide of the disclosure comprising a highaffinity SIRP-α D1 variant binds with higher binding affinity to CD47than a wild-type human SIRP-α D1 domain. In some embodiments, the highaffinity SIRP-α D1 variant binds to human CD47 with at least 1-fold(e.g., at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold,5-fold or greater than 5-fold) affinity than the affinity of a naturallyoccurring D1 domain. In some embodiments, the high affinity SIRP-α D1variant binds to human CD47 with at least 1-fold (e.g., at least10-fold, 100-fold, 1000-fold or greater than 1000-fold) affinity thanthe affinity of a naturally occurring D1 domain.

As used herein, the term “optimized affinity” or “optimized bindingaffinity” refers to an optimized strength of the binding interactionbetween a polypeptide disclosed herein, including a high affinity SIRP-αD1 variant, and CD47. For example, in some embodiments, the polypeptidebinds primarily or with higher affinity to CD47 on cancer cells and doesnot substantially bind or binds with lower affinity to CD47 onnon-cancer cells. In some embodiments, the binding affinity between thepolypeptide and CD47 is optimized such that the interaction does notcause clinically relevant toxicity or decreases toxicity compared to avariant which binds with maximal affinity. In some embodiments, in orderto achieve an optimized binding affinity between a polypeptide providedherein and CD47, the polypeptide including a high affinity SIRP-α D1variant is developed to have a lower binding affinity to CD47 than whichis maximally achievable. In some embodiments, the high affinity SIRP-αvariants disclosed herein cross react with rodent, non-human primate(NHP), and human CD47.

As used herein, the term “immunogenicity” refers to the property of aprotein (e.g., a therapeutic protein) which causes an immune response inthe host as though it is a foreign antigen. The immunogenicity of aprotein can be assayed in vitro in a variety of different ways, such asthrough in vitro T-cell proliferation assays.

As used herein, the term “minimal immunogenicity” refers to animmunogenicity of a protein (e.g., a therapeutic protein) that has beenmodified, e.g., through amino acid substitutions, to be lower (e.g., atleast 10%, 25%, 50%, or 100% lower) than the immunogenicity before theamino acid substitutions are introduced (e.g., an unmodified protein).In some embodiments, a protein (e.g., a therapeutic protein) is modifiedto have minimal immunogenicity and causes no or very little host immuneresponse even though it is a foreign antigen.

In some embodiments, the high affinity SIRP-α D1 variant has minimalimmunogenicity. In some embodiments, a SIRP-α polypeptide of thedisclosure administered to a subject has the same amino acid sequence asthat of the SIRP-α polypeptide in a biological sample of the subject,except for amino acid changes which increase affinity of the SIRP-α D1variant. In some embodiments, the polypeptide variants disclosed hereinlower the risk of side effects compared to anti-CD47 antibodies orwild-type SIRP-α. In some embodiments, the polypeptide variantsdisclosed herein lower the risk of anemia compared to anti-CD47antibodies or wild-type SIRP-α. In some embodiments, the polypeptidevariants disclosed herein do not cause acute anemia in rodent ornon-human primates (NHP) studies.

Table 2 lists specific amino acid substitutions in a high affinitySIRP-α D1 variant relative to each D1 domain sequence. In someembodiments, a high affinity SIRP-α D1 variant includes one or more(e.g., two, three, four, five, six, seven, eight, nine, ten, eleven,twelve, thirteen, fourteen or more) of the substitutions listed in Table2. In some embodiments, a high affinity SIRP-α D1 variant includes atmost fourteen amino acid substitutions relative to a wild-type D1domain. In some embodiments, a high affinity SIRP-α D1 variant includesat most ten amino acid substitutions relative to a wild-type D1 domain.In some embodiments, a high affinity SIRP-α D1 variant includes at mostseven amino acid substitutions relative to a wild-type D1 domain. Insome embodiments, a high affinity SIRP-α D1 variant of the disclosurehas at least 90% (e.g., at least 92%, 95%, 97% or greater than 97%)amino acid sequence identity to a sequence of a wild-type D1 domain.

In some embodiments, a high affinity SIRP-α D1 variant is a chimerichigh affinity SIRP-α D1 variant that includes a portion of two or morewild-type D1 domains or variants thereof (e.g., a portion of onewild-type D1 domain or variant thereof and a portion of anotherwild-type D1 domain or variant thereof). In some embodiments, a chimerichigh affinity SIRP-α D1 variant includes at least two portions (e.g.,three, four, five or more portions) of wild-type D1 domains or variantsthereof, wherein each of the portions is from a different wild-type D1domain. In some embodiments, a chimeric high affinity SIRP-α D1 variantfurther includes one or more amino acid substitutions listed in Table 2.

TABLE 2 Amino Acid Substitutions in a High Affinity SIRP-α D1 VariantSEQ ID NO: Description Amino Acid Sequence 13 D1 domain v1EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRIGNITPADAGTYYCX₁₂KX₁₃RKGS PDDVEX₁₄KSGAGTELSVRAKPS —Amino acid X₁ = L, I, V; X₂ = V, L, I;  substitutionsX₃ = A, V; X₄ = A, I, L; relative to X₅ = I, T, S, F; X₆ = E, V, L; SEQ ID NO: 13 X₇ = K, R; X₈ = E, Q; X₉ = H, P, R; X₁₀ = L, T, G;X₁₁ = K, R; X₁₂ = V, I;  X₁₃ = F, L, V; X₁₄ = F, V 14 D1 domain v2EEEX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISNITPADAGTYYCX₁₂KX₁₃RKGSPD TEX₁₄KSGAGTELSVRAKPS —Amino acid X₁ = L, I, V; X₂ = V, L, I; substitutionsX₃ = A, V; X₄ = V, I, L;  relative to X₅ = I, T, S, F; X₆ = E, V, L; SEQ ID NO: 14 X₇ = K, R; X₈ = E, Q; X₉ = H, P, R; X₁₀ = S, T, G; X₁₁ = K, R; X₁₂ = V, I;  X₁₃ = F, L, V; X₁₄ = F, V 15 D1 domain v3EEEX₁QX₂IQPDKSVSVAAGESX₃ILLCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISNITPADAGTYYCX₁₂KX₁₃RKGSPD TEX₁₄KSGAGTELSVRAKPS —Amino acid X₁ = L, I, V; X₂ = V, L, I; substitutionsX₃ = A, V; X₄ = V, I, L;  relative to X₅ = I, T, S, F; X₆ = E, V, L;SEQ ID NO: 15 X₇ = K, R; X₈ = E, Q;  X₉ = H, P, R; X₁₀ = S, T, G; X₁₁ = K, R; X₁₂ = V, I;  X₁₃ = F, L, V; X₁₄ = F, V 16 D1 domain v4EEGX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRIGNITPADAGTYYCX₁₂KX₁₃RKGSP DDVEX₁₄KSGAGTELSVRAKPS —Amino acid X₁ = L, I, V; X₂ = V, L, I; substitutionsX₃ = A, V; X₄ = A, I, L;  relative to X₅ = I, T, S, F; X₆ = E, V, L; SEQ ID NO: 16 X₇ = K, R; X₈ = E, Q; X₉ = H, P, R; X₁₀ = L, T, G;X₁₁ = K, R; X₁₂ = V, I; X₁₃ = F, L, V; X₁₄ = F, V 17 D1 domain v5EEEX₁QX₂IQPDKFVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRIGNITPADAGTYYCX₁₂KX₁₃RKGS PDDVEX₁₄KSGAGTELSVRAKPS —Amino acid X₁ = L, I, V; X₂ = V, L, I; substitutionsX₃ = A, V; X₄ = A, I, L; relative to X₅ = I, T, S, F; X₆ = E, V, L; SEQ ID NO: 17 X₇ = K, R; X₈ = E, Q;  X₉ = H, P, R; X₁₀ = L, T, G; X₁₁ = K, R; X₁₂ = V, I;  X₁₃ = F, L, V; X₁₄ = F, V 18 D1 domain v6EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFPIRIGNITPADAGTYYCX₁₂KX₁₃RKGS PDDVEX₁₄KSGAGTELSVRAKPS —Amino acid X₁ = L, I, V; X₂ = V, L, I; substitutionsX₃ = A, V; X₄ = A, I, L;  relative to X₅ = I, T, S, F; X₆ = E, V, L; SEQ ID NO: 18 X₇ = K, R; X₈ = E, Q;  X₉ = H, P, R; X₁₀ = L, T, G; X₁₁ = K, R; X₁₂ = V, I;  X₁₃ = F, L, V; X₁₄ = F, V 19 D1 domain v7EEEX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISNITPADAGTYYCX₁₂KX₁₃RKGSPD TEX₁₄KSGAGTELSVRGKPS —Amino acid X₁ = L, I, V; X₂ = V, L, I; substitutionsX₃ = A, V; X₄ = V, I, L;  relative to X₅ = I, T, S, F; X₆ = E, V, L; SEQ ID NO: 19 X₇ = K, R; X₈ = E, Q;  X₉ = H, P, R; X₁₀ = S, T, G; X₁₁ = K, R; X₁₂ = V, I;  X₁₃ = F, L, V; X₁₄ = F, V 20 D1 domain v8EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISNITPADAGTYYCX₁₂KX₁₃RKGSP DTEX₁₄KSGAGTELSVRAKPS —Amino acid X₁ = L, I, V; X₂ = V, L, I; substitutionsX₃ = A, V; X₄ = A, I, L;  relative to X₅ = I, T, S, F; X₆ = E, V, L; SEQ ID NO: 20 X₇ = K, R; X₈ = E, Q;  X₉ = H, P, R; X₁₀ = S, T, G; X₁₁ = K, R; X₁₂ = V, I;  X₁₃ = F, L, V; X₁₄ = F, V 21 D1 domain v9EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRISNITPADAGTYYCX₁₂KX₁₃RKGSP DDVEX₁₄KSGAGTELSVRAKPS —Amino acid X₁ = L, I, V; X₂ = V, L, I; substitutionsX₃ = A, V; X₄ = A, I, L;  relative to X₅ = I, T, S, F; X₆ = E, V, L; SEQ ID NO: 21 X₇ = K, R; X₈ = E, Q;  X₉ = H, P, R; X₁₀ = L, T, G; X₁₁ = K, R; X₁₂ = V, I;  X₁₃ = F, L, V; X₁₄ = F, V 22 D1 domain v10EEEX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISNITPADAGTYYCX₁₂KX₁₃RKGSPD TEX₁₄KSGAGTELSVRAKPS —Amino acid X₁ = L, I, V; X₂ = V, L, I; substitutionsX₃ = A, V; X₄ = V, I, L;  relative to X₅ = I, T, S, F; X₆ = E, V, L; SEQ ID NO: 22 X₇ = K, R; X₈ = E, Q;  X₉ = H, P, R; X₁₀ = S, T, G; X₁₁ = K, R; X₁₂ = V, I;  X₁₃ = F, L, V; X₁₄ = F, V 23 Pan D1 domainEEX₁X₂QX₃IQPDKX₄VX₅VAAGEX₆X₇X₈LX₉CTX₁₀TSLX₁₁PVGPIQWFRGAGPX₁₂RX₁₃LIYNQX₁₄X₁₅GX₁₆FPRVTTVSX₁₇X₁₈TX₁₉RX₂₀NMDFX₂₁IX₂₂IX₂₃NITPADAGTYYCX₂₄KX₂₅RKGSPDX₂₆X₂₇EX₂₈KSGAGTELSVRX₂₉ KPS — Amino acidX₁ = E, G; X₂ = L, I, V;   substitutions X₃ = V, L, I; X₄ = S, F;relative to X₅ = L, S; X₆ = 5, T;  SEQ ID NO: 23 X₇ = A, V; X₈ = I, T; X₉ = H, R; X₁₀ = A, V, I, L;  X₁₁ = I, T, S, F; X₁₂ = A, G; X₁₃ = E, V, L; X₁₄ = K, R;  X₁₅ = E, Q; X₁₆ = H, P, R; X₁₇ = D, E; X₁₈ = S, L, T, G;  X₁₉ = K, R; X₂₀ = E, D;X₂₁ = S, P; X₂₂ = S, R;  X₂₃ = S, G; X₂₄ = V, I;X₂₅ = F, L, V; X₂₆ = D or absent;  X₂₇ = T, V; X₂₈ = F, V; and X₂₉ = A, G

In some embodiments, a polypeptide includes a SIRP-α D1 variant having asequence of:EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRIGNITPADAGTYYCX₁₂KX₁₃RKGSPDDVEX₁₄KSGAGTELSVRAKPS (SEQ ID NO: 13), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃is A or V; X₄ is A, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is L, T, or G; X₁₁ is Kor R; X₁₂ is V or I; X₁₃ is F, L, or V; and X₁₄ is F or V; and whereinthe variant has at least one amino acid substitution relative to awild-type SIRP-α D1 domain having the sequence of SEQ ID NO: 1.

In some embodiments, a polypeptide includes a SIRP-α D1 variant having asequence of:EEGX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRIGNITPADAGTYYCX₁₂KX₁₃RKGSPDDVEX₁₄KSGAGTELSVRAKPS (SEQ ID NO: 16), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃is A or V; X₄ is A, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is L, T, or G; X₁₁ is Kor R; X₁₂ is V or I; X₁₃ is F, L, or V; and X₁₄ is F or V; and whereinthe variant has at least one amino acid substitution relative to awild-type SIRP-α D1 domain having the sequence of SEQ ID NO: 4.

In some embodiments, a polypeptide includes a SIRP-α D1 variant having asequence of:EEEX₁QX₂IQPDKFVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRIGNITPADAGTYYCX₁₂KX₁₃RKGSPDDVEX₁₄KSGAGTELSVRAKPS (SEQ ID NO: 17), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃is A or V; X₄ is A, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is L, T, or G; X₁₁ is Kor R; X₁₂ is V or I; X₁₃ is F, L, or V; and X₁₄ is F or V; and whereinthe variant has at least one amino acid substitution relative to awild-type SIRP-α D1 domain having the sequence of SEQ ID NO: 5.

In some embodiments, a polypeptide includes a SIRP-α D1 variant having asequence of:EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFPIRIGNITPADAGTYYCX₁₂KX₁₃RKGSPDDVEX₁₄KSGAGTELSVRAKPS (SEQ ID NO: 18), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃is A or V; X₄ is A, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is L, T, or G; X₁₁ is Kor R; X₁₂ is V or I; X₁₃ is F, L, or V; and X₁₄ is F or V; and whereinthe variant has at least one amino acid substitution relative to awild-type SIRP-α D1 domain having the sequence of SEQ ID NO: 6.

In some embodiments, a polypeptide includes a SIRP-α D1 variant having asequence of:EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRISNITPADAGTYYCX₁₂KX₁₃RKGSPDDVEX₁₄KSGAGTELSVRAKPS (SEQ ID NO: 21), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃is A or V; X₄ is A, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is L, T, or G; X₁₁ is Kor R; X₁₂ is V or I; X₁₃ is F, L, or V; and X₁₄ is F or V; and whereinthe variant has at least one amino acid substitution relative to awild-type SIRP-α D1 domain having the sequence of SEQ ID NO: 9.

In any of the aforementioned embodiments, a polypeptide includes a highaffinity SIRP-α D1 variant having a sequence of any one of SEQ ID NOs:13, 16-18, and 21, wherein X₁ is L, I, or V. In any of theaforementioned embodiments, X₂ is V, L, or, I. In any of theaforementioned embodiments, X₃ is A or V. In any of the aforementionedembodiments, X₄ is A, I, or L. In any of the aforementioned embodiments,X₅ is I, T, S, or F. In any of the aforementioned embodiments, X₆ is E,V, or L. In any of the aforementioned embodiments, X₇ is K or R. In anyof the aforementioned embodiments, X₈ is E or Q. In any of theaforementioned embodiments, X₉ is H, P, or R. In any of theaforementioned embodiments, X₁₀ is L, T, or G. In any of theaforementioned embodiments, X₁₁ is K or R. In any of the aforementionedembodiments, X₁₂ is V or I. In any of the aforementioned embodiments,X₁₃ is F, L, V. In any of the aforementioned embodiments, X₁₄ is F or V.In some embodiments, the polypeptide of this aspect of the disclosureincludes no more than six amino acid substitutions relative to thewild-type SIRP-α D1 domain having the sequence of any one of SEQ ID NOs:1, 4-6, and 9.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRP-α D1 domain having thesequence of any one of SEQ ID NOs: 1, 4-6, and 9. In some embodiments,the polypeptide binds CD47 with at least 100-fold greater bindingaffinity than the wild-type SIRP-α D1 domain having the sequence of anyone of SEQ ID NOs: 1, 4-6, and 9. In some embodiments, the polypeptidebinds CD47 with at least 1000-fold greater binding affinity than thewild-type SIRP-α D1 domain having the sequence of any one of SEQ ID NOs:1, 4-6, and 9. In some embodiments, a SIRP-α D1 variant polypeptide orfragment thereof binds to CD47 with a KD less than 1×10⁻⁸ M, less than5×10⁻⁹ M, less than 1×10⁻⁹ M, less 5×10⁻¹⁰ M, less than 1×10⁻¹⁰ M orless than 1×10⁻¹¹ M. In some embodiments, a SIRP-α D1 variantpolypeptide or fragment thereof binds to CD47 with a KD between about500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nMand 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM,between about 1 nM and 500 pM, between about 500 pM and 100 pM, betweenabout 100 pM and 50 pM, or between about 50 pM and 10 pM.

In some embodiments, a polypeptide includes a SIRP-α D1 variant having asequence of:EEEX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISNITPADAGTYYCX₁₂KX₁₃RKGSPDTEX₁₄KSGAGTELSVR AKPS(SEQ ID NO: 14), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃ is A orV; X₄ is V, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇ is K orR; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is S, T, or G; X₁₁ is K or R; X₁₂is V or I; X₁₃ is F, L, or V; and X₁₄ is F or V; and wherein the varianthas at least one amino acid substitution relative to a wild-type SIRP-αD1 domain having the sequence of SEQ ID NO: 2.

In some embodiments, a polypeptide includes a SIRP-α D1 variant having asequence of:EEEX₁QX₂IQPDKSVSVAAGESX₃ILLCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISNITPADAGTYYCX₁₂KX₁₃RKGSPDTEX₁₄KSGAGTELSVRA KPS(SEQ ID NO: 15), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃ is A orV; X₄ is V, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇ is K orR; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is S, T, or G; X₁₁ is K or R; X₁₂is V or I; X₁₃ is F, L, or V; and X₁₄ is F or V; and wherein the varianthas at least one amino acid substitution relative to a wild-type SIRP-αD1 domain having the sequence of SEQ ID NO: 3.

In some embodiments, a polypeptide includes a SIRP-α D1 variant having asequence of:EEEX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISNITPADAGTYYCX₁₂KX₁₃RKGSPDTEX₁₄KSGAGTELSVR GKPS(SEQ ID NO: 19), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃ is A orV; X₄ is V, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇ is K orR; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is S, T, or G; X₁₁ is K or R; X₁₂is V or I; X₁₃ is F, L, or V; and X₁₄ is F or V; and wherein the varianthas at least one amino acid substitution relative to a wild-type SIRP-αD1 domain having the sequence of SEQ ID NO: 7.

In some embodiments, a polypeptide includes a SIRP-α D1 variant having asequence of:EEEX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISNITPADAGTYYCX₁₂KX₁₃RKGSPDTEX₁₄KSGAGTELSVR AKPS(SEQ ID NO: 22), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃ is A orV; X₄ is V, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇ is K orR; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is S, T, or G; X₁₁ is K or R; X₁₂is V or I; X₁₃ is F, L, or V; and X₁₄ is F or V; and wherein the varianthas at least one amino acid substitution relative to a wild-type SIRP-αD1 domain having the sequence of SEQ ID NO: 10.

In any of the aforementioned embodiments in this aspect of thedisclosure, the polypeptide has the sequence of any one of SEQ ID NOs:14, 15, 19, and 22, wherein X₁ is L, I, or V. In any of theaforementioned embodiments, X₂ is V, L, or, I. In any of theaforementioned embodiments, X₃ is A or V. In any of the aforementionedembodiments, X₄ is V, I, or L. In any of the aforementioned embodiments,X₅ is I, T, S, or F. In any of the aforementioned embodiments, X₆ is E,V, or L. In any of the aforementioned embodiments, X₇ is K or R. In anyof the aforementioned embodiments, X₈ is E or Q. In any of theaforementioned embodiments, X₉ is H, P, or R. In any of theaforementioned embodiments, X₁₀ is S, T, or G. In any of theaforementioned embodiments, X₁₁ is K or R. In any of the aforementionedembodiments, X₁₂ is V or I. In any of the aforementioned embodiments,X₁₃ is F, L, or V. In any of the aforementioned embodiments, X₁₄ is F orV. In some embodiments, the polypeptide of this aspect of the disclosureincludes no more than six amino acid substitutions relative to thewild-type SIRP-α D1 domain having the sequence of any one of SEQ ID NOs:2, 3, 7, and 10.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRP-α D1 domain having thesequence of any one of SEQ ID NOs: 2, 3, 7, and 10. In some embodiments,the polypeptide binds CD47 with at least 100-fold greater bindingaffinity than the wild-type SIRP-α D1 domain having the sequence of anyone of SEQ ID NOs: 2, 3, 7, and 10. In some embodiments, the polypeptidebinds CD47 with at least 1000-fold greater binding affinity than thewild-type SIRP-α D1 domain having the sequence of any one of SEQ ID NOs:2, 3, 7, and 10. In some embodiments, a SIRP-α D1 variant polypeptide orfragment thereof binds to CD47 with a K_(D) less than 1×10⁻⁸ M, lessthan 5×10⁻⁹ M, less than 1×10⁻⁹ M, less 5×10⁻¹⁰ M, less than 1×10⁻¹⁰M orless than 1×10⁻¹¹M. In some embodiments, a SIRP-α D1 variant polypeptideor fragment thereof binds to CD47 with a K_(D) between about 500 nM and100 nM, between about 100 nM and 50 nM, between about 50 nM and 10 nM,between about 10 nM and 5 nM, between about 5 nM and 1 nM, between about1 nM and 500 pM, between about 500 pM and 100 pM, between about 100 pMand 50 pM, or between about 50 pM and 10 pM.

In some embodiments, a polypeptide includes a SIRP-α D1 variant having asequence of:EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISNITPADAGTYYCX₁₂KX₁₃RKGSPDTEX₁₄KSGAGTELSVR AKPS(SEQ ID NO: 20), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃ is A orV; X₄ is A, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇ is K orR; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is S, T, or G; X₁₁ is K or R; X₁₂is V or I; X₁₃ is F, L, or V; and X₁₄ is F or V; and wherein the varianthas at least one amino acid substitution relative to a wild-type SIRP-αD1 domain having the sequence of SEQ ID NO: 8.

In some embodiments, the polypeptide has the sequence of SEQ ID NO: 20,wherein X₁ is L, I, or V. In any of the aforementioned embodiments inthis aspect of the disclosure, X₂ is V, L, or, I. In any of theaforementioned embodiments, X₃ is A or V. In any of the aforementionedembodiments, X₄ is A, I, or L. In any of the aforementioned embodiments,X₅ is I, T, S, or F. In any of the aforementioned embodiments, X₆ is E,V, or L. In any of the aforementioned embodiments, X₇ is K or R. In anyof the aforementioned embodiments, X₈ is E or Q. In any of theaforementioned embodiments, X₉ is H, P, or R. In any of theaforementioned embodiments, X₁₀ is S, T, or G. In any of theaforementioned embodiments, X₁₁ is K or R. In any of the aforementionedembodiments, X₁₂ is V or I. In any of the aforementioned embodiments,X₁₃ is F, L, or V. In any of the aforementioned embodiments, X₁₄ is F orV. In some embodiments, the polypeptide of this aspect of the disclosureincludes no more than six amino acid substitutions relative to thewild-type SIRP-α D1 domain having the sequence of SEQ ID NO: 8.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRP-α D1 domain having thesequence of SEQ ID NO: 8. In some embodiments, the polypeptide bindsCD47 with at least 100-fold greater binding affinity than the wild-typeSIRP-α D1 domain having the sequence of SEQ ID NO: 8. In someembodiments, the polypeptide binds CD47 with at least 1000-fold greaterbinding affinity than the wild-type SIRP-α D1 domain having the sequenceof SEQ ID NO: 8. In some embodiments, a SIRP-α D1 variant polypeptide orfragment thereof binds to CD47 with a K_(D) less than 1×10⁻⁸ M, lessthan 5×10⁻⁹ M, less than 1×10⁻⁹ M, less 5×10⁻¹⁰ M, less than 1×10⁻¹⁰ Mor less than 1×10⁻¹¹M. In some embodiments, a SIRP-α D1 variantpolypeptide or fragment thereof binds to CD47 with a K_(D) between about500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nMand 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM,between about 1 nM and 500 pM, between about 500 pM and 100 pM, betweenabout 100 pM and 50 pM, or between about 50 pM and 10 pM.

In some embodiments, a polypeptide includes a SIRP-α D1 variant having asequence of:EEX₁X₂QX₃IQPDKX₄VX₅VAAGEX₆X₇X₈LX₉CTX₁₀TSLX₁₁PVGPIQWFRGAGPX₁₂RX₁₃LIYNQX₁₄X₁₅GX₁₆FPRVTTVSX₁₇X₁₈TX₁₉RX₂₀NMDFX₂₁IX₂₂IX₂₃NITPADAGTYYCX₂₄KX₂₅RKGSPDX₂₆X₂₇EX₂₈KSGAGTELSVRX₂₉KPS(SEQ ID NO: 23), wherein X₁ is E or G; X₂ is L, I, or V; X₃ is V, L, or,I; X₄ is S or F; X₅ is L or S; X₆ is S or T; X₇ is A or V; X₈ is I or T;X₉ is H or R; X₁₀ is A, V, I, or L; X₁₁ is I, T, S, or F; X₁₂ is A or G;X₁₃ is E, V, or L; X₁₄ is K or R; X₁₅ is E or Q; X₁₆ is H, P, or R; X₁₇is D or E; X₁₈ is S, L, T, or G; X₁₉ is K or R; X₂₀ is E or D; X₂₁ is Sor P; X₂₂ is S or R; X₂₃ is S or G; X₂₄ is V or I; X₂₅ is F, L, V; X₂₆is D or absent; X₂₇ is T or V; X₂₈ is F or V; and X₂₉ is A or G; andwherein the variant has at least one amino acid substitution relative toa wild-type SIRP-α D1 domain having the sequence of any one of SEQ IDNOs: 1-10.

In any of the aforementioned embodiments in this aspect of thedisclosure, X₂ is L, I, or V. In any of the aforementioned embodiments,X₃ is V, L, or, I. In any of the aforementioned embodiments, X₄ is S orF. In any of the aforementioned embodiments, X₅ is L or S. In any of theaforementioned embodiments, X₆ is S or T. In any of the aforementionedembodiments, X₇ is A or V. In any of the aforementioned embodiments, X₈is I or T. In any of the aforementioned embodiments, X₉ is H or R. Inany of the aforementioned embodiments, X₁₀ is A, V, I, or L. In any ofthe aforementioned embodiments, X₁₁ is I, T, S, or F. In any of theaforementioned embodiments, X₁₂ is A or G. In any of the aforementionedembodiments, X₁₃ is E, V, or L. In any of the aforementionedembodiments, X₁₄ is K or R. In any of the aforementioned embodiments,X₁₅ is E or Q. In any of the aforementioned embodiments, X₁₆ is H, P, orR. In any of the aforementioned embodiments, X₁₇ is D or E. In any ofthe aforementioned embodiments, X₁₈ is S, L, T, or G. In any of theaforementioned embodiments, X₁₉ is K or R. In any of the aforementionedembodiments, X₂₀ is E or D. In any of the aforementioned embodiments,X₂₁ is S or P. In any of the aforementioned embodiments, X₂₂ is S or R.In any of the aforementioned embodiments, X₂₃ is S or G. In any of theaforementioned embodiments, X₂₄ is V or I. In any of the aforementionedembodiments, X₂₅ is F, L, V. In any of the aforementioned embodiments,X₂₆ is D or absent. In any of the aforementioned embodiments, X₂₇ is Tor V. In any of the aforementioned embodiments, X₂₈ is F or V. In any ofthe aforementioned embodiments, X₂₉ is A or G. In some embodiments, thepolypeptide of this aspect of the disclosure includes no more than sixamino acid substitutions relative to the wild-type SIRP-α D1 domainhaving the sequence of any one of SEQ ID NOs: 1-10.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRP-α D1 domain having thesequence of any one of SEQ ID NOs: 1-10. In some embodiments, thepolypeptide binds CD47 with at least 100-fold greater binding affinitythan the wild-type SIRP-α D1 domain having the sequence of any one ofSEQ ID NOs: 1-10. In some embodiments, the polypeptide binds CD47 withat least 1000-fold greater binding affinity than the wild-type SIRP-α D1domain having the sequence of any one of SEQ ID NOs: 1-10. In someembodiments, a SIRP-α D1 variant polypeptide or fragment thereof bindsto CD47 with a K_(D) less than 1×10⁻⁸ M, less than 5×10⁻⁹ M, less than1×10⁻⁹ M, less 5×10⁻¹⁰ M, less than 1×10⁻¹⁰ M or less than 1×10⁻¹¹ M. Insome embodiments, a SIRP-α D1 variant polypeptide or fragment thereofbinds to CD47 with a K_(D) between about 500 nM and 100 nM, betweenabout 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM,between about 500 pM and 100 pM, between about 100 pM and 50 pM, orbetween about 50 pM and 10 pM.

In some embodiments, a polypeptide of the disclosure including a highaffinity SIRP-α D1 variant further comprises a D2 domain having thesequence of SEQ ID NO: 24, a D3 domain having the sequence of SEQ ID NO:25, or a D2 domain having the sequence of SEQ ID NO: 24 and a D3 domainhaving the sequence of SEQ ID NO: 25 of a wild-type human SIRP-α asshown in Table 3. In some embodiments, the high affinity SIRP-α D1variant further comprises a fragment or variant of a D2 domain or afragment or variant of a D3 domain. In some embodiments, the highaffinity SIRP-α D1 variant further comprises a fragment or variant of aD2 domain and a fragment or variant of a D3 domain. In some embodiments,a high affinity SIRP-α D1 variant is joined to a D2 or D3 domain by wayof a linker. In some embodiments, a high affinity SIRP-α D1 variant isjoined to a D2 and D3 domain by way of a linker.

TABLE 3 Amino Acid Sequences of SIRP-α D2 and D3 Domains SEQ ID NO:Description Amino Acid Sequence 24 SIRP-α D2 APVVSGPAARATPQHTVSFTCESHdomain GFSPRDITLKWFKNGNELSDFQTN VDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTAN LSETIR 25 SIRP-α D3 VPPTLEVTQQPVRAENQVNVTCQVdomain RKFYPQRLQLTWLENGNVSRTETA STVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDL KVS

In some embodiments, a polypeptide of the disclosure including a highaffinity SIRP-α D1 variant is attached to an Fc domain monomer, a humanserum albumin (HSA) or variant thereof, a serum-binding protein orpeptide, or an organic molecule, e.g., a polymer (e.g., a PEG polymer),in order to improve the pharmacokinetic properties of the polypeptide,e.g., increase serum half-life. In some embodiments, a high affinitySIRP-α D1 variant is attached to an Fc domain monomer that is unable todimerize. In some embodiments, Fc domain monomers, HSA proteins,serum-binding proteins or peptides, and organic molecules such as a PEGserve to increase the serum half-life of the polypeptides describedherein. In some embodiments, a polypeptide of the disclosure including ahigh affinity SIRP-α D1 variant does not include the sequence of any oneof SEQ ID NOs: 26-36 shown in Table 4.

TABLE 4 SEQ ID NO: Amino Acid Sequence 26EEELQVIQPDKSVSVAAGESAILHCTITSLIPVGPIQWFRGAGPARELIYNQREGHFPRVTTVSETTRRENMDFSISISNITPADAGTYYCVKFRKGSPDTEVKSGAGTELSVRAKPS 27EEEVQVIQPDKSVSVAAGESAILHCTLTSLIPVGPIQWFRGAGPARVLIYNQRQGHFPRVTTVSEGTRRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGAGTELSVRAKPS 28EEEVQIIQPDKSVSVAAGESVILHCTITSLTPVGPIQWFRGAGPARLLIYNQREGPFPRVTTVSETTRRENMDFSISISNITPADAGTYYCVKLRKGSPDTEFKSGAGTELSVRAKPS 29EEELQIIQPDKSVSVAAGESAILHCTITSLSPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTVSEGTKRENMDFSISISNITPADAGTYYCIKLRKGSPDTEFKSGAGTELSVRAKPS 30EEEIQVIQPDKSVSVAAGESVIIHCTVTSLFPVGPIQWFRGAGPARVLIYNQRQGRFPRVTTVSEGTKRENMDFSISISNITPADAGTYYCVKVRKGSPDTEVKSGAGTELSVRAKPS 31EEEVQIIQPDKSVSVAAGESIILHCTVTSLFPVGPIQWFRGAGPARVLIYNQREGRFPRVTTVSEGTRRENMDFSISISNITPADAGTYYCIKLRKGSPDTEFKSGAGTELSVRAKPS 32EEEVQLIQPDKSVSVAAGESAILHCTVTSLFPVGPIQWFRGAGPARVLIYNQREGPFPRVTTVSEGTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEVKSGAGTELSVRAKPS 33EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPS 34EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARLLIYNQRQGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS 35EEEVQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQKQGPFPRVTTISETTRRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGAGTELSVRAKPS 36EEELQIIQPDKSVSVAAGESAILHCTITSLTPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTVSEGTRRENMDFSISISNITPADAGTYYCIKFRKGSPDTEVKSGAGTELSVRAKPS

In some embodiments, the polypeptides and polypeptide constructsdescribed herein are utilized in vitro for binding assays, such asimmune assays. For example, in some embodiments, the polypeptides andpolypeptide constructs described herein are utilized in liquid phase orbound to a solid phase carrier. In some embodiments, polypeptidesutilized for immunoassays are detectably labeled in various ways.

In some embodiments, polypeptides and polypeptide constructs describedherein are bound to various carriers and used to detect the presence ofspecific antigen expressing cells. Examples of carriers include glass,polystyrene, polypropylene, polyethylene, dextran, nylon, amylases,natural and modified celluloses, polyacrylamides, agaroses, andmagnetite. The nature of the carrier can be either soluble or insoluble.

Various different labels and methods of labeling are known. Examples oflabels include enzymes, radioisotopes, fluorescent compounds, colloidalmetals, chemiluminescent compounds, and bio-luminescent compounds.Various techniques for binding labels to polypeptides disclosed hereinare available.

In some embodiments, the polypeptides are coupled to low molecularweight haptens. These haptens are then specifically detected by means ofa second reaction. For example, in some embodiments, the hapten biotinis used with avidin or the haptens dinitrophenol, pyridoxal, orfluorescein are detected with specific anti-hapten antibodies (e.g.,anti-dinitrophenol antibodies, anti-pyridoxal antibodies, andanti-fluorescein antibodies respectively).

II. High Affinity SIRP-α D1 Domains with Altered Glycosylation

Disclosed herein, in some embodiments, are polypeptides comprising asignal-regulatory protein α (SIRP-α) D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92.

Also disclosed herein, in some embodiments, are polypeptides comprisingan Fc variant, wherein the Fc variant comprises an Fc domain dimerhaving two Fc domain monomers, wherein each Fc domain monomerindependently is selected from (i) a human IgG1 Fc region consisting ofmutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc regionconsisting of mutations A330S, P331S and N297A; or (iii) a human IgG4 Fcregion comprising mutations S228P, E233P, F234V, L235A, delG236, andN297A.

In some embodiments, a polypeptide in a composition disclosed hereincomprises a high affinity SIRP-α D1 variant that has reduced or minimalglycosylation. The D1 domain of each of the ten wild-type human SIRP-αproteins (SEQ ID NOs: 1-10 in Table 1) contains a single potentialN-linked glycosylation site at amino acid N80 in the sequence N80ITP.Expression of a SIRP-α D1 domain in Chinese Hamster Ovary (CHO) cellsresults in a major band of 16 kDa (non-glycosylated) and a minor band ofhigher molecular weight that was removed by Endo Hf. Endo Hf is arecombinant protein fusion of Endoglycosidase H and maltose bindingprotein. Endo Hf cleaves within the chitobiose core of high mannose andsome hybrid oligosaccharides from N-linked glycoproteins. This impliesthat a proline at amino acid position 83 can reduce the efficiency ofglycosylation, leading to a protein with different degrees ofglycosylation and therefore heterogeneity. For drug development,heterogeneity can give rise to challenges in process development.Therefore, to investigate the possibility of generating homogenous,non-glycosylated forms of high affinity SIPR-α D1 variants, in someembodiments, amino acid N80 of a SIPR-α D1 variant is mutated to Ala. Insome embodiments, to make a non-glycosylated, high affinity SIRP-α D1variant, amino acid N80 in a high affinity SIRP-α D1 variant is replacedby any amino acid, including any naturally and non-naturally occurringamino acid, e.g., N80A and N80Q. In some embodiments, a high affinitySIRP-α D1 variant comprises an N80A mutation and at least 1 additionalmutation (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 additionalmutations or more). In some embodiments, the additional mutation is inthe CD47 binding site. In some embodiments, the additional mutation isin the hydrophobic core of the D1 domain.

In some embodiments, a polypeptide in a composition disclosed hereinincludes a high affinity SIRP-α D1 variant that has increasedglycosylation relative to a wild-type SIRP-α D1 domain. Another optionto increase homogeneity of the final product is to enhance theefficiency of glycosylation at amino acid N80 and generate high affinitySIRP-α D1 variants with increased glycosylation relative to a wild-type.In some embodiments, the amino acid P83 in the sequence NITP83 affectsthe degree of glycosylation at amino acid N80. In some embodiments,changing P83 to any amino acid increases the efficiency of glycosylationat N80. In some embodiments, amino acid P83 in a high affinity SIRP-α D1variant is replaced by any amino acid, including naturally andnon-naturally amino acids, e.g., P83V, P83A, P83I, and P83L. In someembodiments, a polypeptide of the disclosure is expressed in a cell thatis optimized not to glycosylate proteins that are expressed by suchcell, for example by genetic engineering of the cell line (e.g.,genetically engineered yeast or mammalian host) or modifications of cellculture conditions such as addition of kifunensine or by using anaturally non-glycosylating host such as a prokaryote (E. coli, etc.).

Table 5 lists specific amino acid substitutions in a high affinitySIRP-α D1 variant relative to each D1 domain variant sequence. In someembodiments, a high affinity SIRP-α D1 variant includes one or more(e.g., two, three, four, five, six, seven, eight, nine, ten, eleven,twelve, thirteen, fourteen or more) of the substitutions listed in Table5. In some embodiments, the SIRP-α D1 variants are not glycosylated orare minimally glycosylated. In some embodiments, the SIRP-α D1 variantsare fully glycosylated or almost fully glycosylated. In someembodiments, a high affinity SIRP-α D1 variant includes at most fourteenamino acid substitutions relative to a wild-type D1 domain. In someembodiments, a high affinity SIRP-α D1 variant includes at most tenamino acid substitutions relative to a wild-type D1 domain. In someembodiments, a high affinity SIRP-α D1 variant includes at most sevenamino acid substitutions relative to a wild-type D1 domain. In someembodiments, a high affinity SIRP-α D1 variant of the disclosure has atleast 90% (e.g., at least 92%, 95%, 97% or greater than 97%) amino acidsequence identity to a sequence of a wild-type D1 domain.

In some embodiments, a high affinity SIRP-α D1 variant is a chimerichigh affinity SIRP-α D1 variant that includes a portion of two or morewild-type D1 domains or variants thereof (e.g., a portion of onewild-type D1 domain or variant thereof and a portion of anotherwild-type D1 domain or variant thereof). In some embodiments, a chimerichigh affinity SIRP-α D1 variant includes at least two portions (e.g.,three, four, five or more portions) of wild-type D1 domains or variantsthereof, wherein each of the portions is from a different wild-type D1domain. In some embodiments, a chimeric high affinity SIRP-α D1 variantfurther includes one or more amino acid substitutions listed in Table 5.

TABLE 5 Amino Acid Substitutions in a High Affinity SIRP-α D1 VariantSEQ ID NO: Description Amino Acid Sequence  37 D1 domain v1EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PV GPIQWFRGAGPGRX₆LIYNQX₇TX₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRIGX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RK  GSPDDVEX₁₆KSGAGTELSVRAKPS —Amino acid X₁ = L, I, V; X₂ = V, L, I;  substitutionsX₃ = A, V; X₄ = A, I, L;  relative to X₅ = I, T, S, F; X₆ = E, V, L; SEQ ID NO: 37 X₇ = K, R; X₈ = E, Q;  X₉ = H, P, R; X₁₀ = L, T, G; X₁₁ = K, R; X₁₂ = N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, Y; X₁₃ = P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y;  X₁₄ = V, I; X₁₅ = F, L, V; X₁₆ = F, V  38 D1 domain v2 EEEX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVG PIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀T X₁₁RENMDFSISISX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGS  PDTEX₁₆KSGAGTELSVRAKPS —Amino acid X₁ = L, I, V; X₂ = V, L, I;  substitutionsX₃ = A, V; X₄ = V, I, L;  relative to X₅ = I, T, S, F; X₆ = E, V, L; SEQ ID NO: 38 X₇ = K, R; X₈ = E, Q;  X₉ = H, P, R; X₁₀ = S, T, G; X₁₁ = K, R; X₁₂ = N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, Y; X₁₃ = P, A, C, D, E, F, G, H, I,  K, L, M, N, Q, R, S, T, V, W, Y; X₁₄ = V, I; X₁₅ = F, L, V; X₁₆ = F, V  39 D1 domain v3EEEX₁QX₂IQPDKSVSVAAGESX₃ILLCTX4TSLX₅PVG PIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀T X₁₁RENMDFSISISX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGS  PDTEX₁₆KSGAGTELSVRAKPS —Amino acid X₁ = L, I, V; X₂ = V, L, I;  substitutionsX₃ = A, V; X₄ = V, I, L;  relative to X₅ = I, T, S, F; X₆ = E, V, L; SEQ ID NO: 39 X₇ = K, R; X₈ = E, Q;  X₉ = H, P, R; X₁₀ = S, T, G; X₁₁ = K, R; X₁₂ = N, A, C, D, E, F, G,H, I, K, L, M, P, Q, R, S, T, V, W, Y; X₁₃ = P, A, C, D, E, F, G, H, I, K, L,  M, N, Q, R, S, T, V, W, Y; X₁₄ = V, I; X₁₅ = F, L, V; X₁₆ = F, V  40 D1 domain v4EEGX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVG PIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀ TX₁₁RNNMDFSIRIGX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGS PDDVEX₁₆KSGAGTELSVRAKPS —Amino acid X₁ = L, I, V; X₂ = V, L, I;  substitutionsX₃ = A, V; X₄ = A, I, L;  relative to X₅ = I, T, S, F; X₆ = E, V, L; SEQ ID NO: 40 X₇ = K, R; X₈ = E, Q; X₉ = H, P, R; X₁₀ = L, T, G; X₁₁ = K, R;  X₁₂ = N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, Y;  X₁₃ = P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y;  X₁₄ = V, I; X₁₅ = F, L, V; X₁₆ = F, V 41 D1 domain v5 EEEX₁QX₂IQPDKFVLVAAGETX₃TLRCTX₄TSLX₅PV GPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀ TX₁₁RNNMDFSIRIGX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RK  GSPDDVEX₁₆KSGAGTELSVRAKPS —Amino acid X₁ = L, I, V; X₂ = V, L, I; X₃ = A, V;  substitutionsX₄ = A, I, L; X₅ = I, T, S, F;  relative toX₆ = E, V, L; X₇ = K, R; X₈ = E, Q;  SEQ ID NO: 41X₉ = H, P, R; X₁₀ = L, T, G;  X₁₁ = K, R; X₁₂ = N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S,  T, V, W, Y; X₁₃ = P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S,  T, V, W, Y; X₁₄ = V, I; X₁₅ = F, L, V; X₁₆ = F, V  42 D1 domain v6EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PV GPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀ TX₁₁RNNMDFPIRIGX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RK  GSPDDVEX₁₆KSGAGTELSVRAKPS —Amino acid X₁ = L, I, V; X₂ = V, L, I; X₃ = A, V;  substitutionsX₄ = A, I, L; X₅ = I, T, S, F;  relative toX₆ = E, V, L; X₇ = K, R; X₈ = E, Q;  SEQ ID NO: 42X₉ = H, P, R; X₁₀ = L, T, G; X₁₁ = K, R; X₁₂ = N, A, C, D, E, F, G, H, I, K,  L, M, P, Q, R, S, T, V, W, Y; X₁₃ = P, A, C, D, E, F, G, H, I, K,  L, M, N, Q, R, S, T, V, W, Y; X₁₄ = V, I; X₁₅ = F, L, V; X₁₆ = F, V  43 D1 domain v7EEEX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVG PIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀T X₁₁RENMDFSISISX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGS  PDTEX₁₆KSGAGTELSVRGKPS —Amino acid X₁ = L, I, V; X₂ = V, L, I; X₃ = A, V;  substitutionsX₄ = V, I, L; X₅ = I, T, S, F;  relative toX₆ = E, V, L; X₇ = K, R; X₈ = E, Q;  SEQ ID NO: 43X₉ = H, P, R; X₁₀ = S, T, G; X₁₁ = K, R; X₁₂ = N, A, C, D, E, F, G, H, I, K,  L, M, P, Q, R, S, T, V, W, Y; X₁₃ = P, A, C, D, E, F, G, H, I, K,  L, M, N, Q, R, S, T, V, W, Y; X₁₄ = V, I; X₁₅ = F, L, V; X₁₆ = F, V  44 D1 domain v8EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PV GPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀ TX₁₁RENMDFSISISX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RK  GSPDTEX₁₆KSGAGTELSVRAKPS —Amino acid X₁ = L, I, V; X₂ = V, L, I; X₃ = A, V;  substitutionsX₄ = A, I, L; X₅ = I, T, S, F;  relative toX₆ = E, V, L; X₇ = K, R; X₈ = E, Q;  SEQ ID NO: 44X₉ = H, P, R; X₁₀ = S, T, G; X₁₁ = K, R; X₁₂ = N, A, C, D, E, F, G, H, I, K, L,  M, P, Q, R, S, T, V, W, Y; X₁₃ = P, A, C, D, E, F, G, H, I, K, L,  M, N, Q, R, S, T, V, W, Y; X₁₄ = V, I; X₁₅ = F, L, V; X₁₆ = F, V  45 D1 domain v9EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PV GPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀ TX₁₁RNNMDFSIRISX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RK  GSPDDVEX₁₆KSGAGTELSVRAKPS —Amino acid X₁ = L, I, V; X₂ = V, L, I; X₃ = A, V;  substitutionsX₄ = A, I, L; X₅ = I, T, S, F;  relative toX₆ = E, V, L; X₇ = K, R; X₈ = E, Q;  SEQ ID NO: 45X₉ = H, P, R; X₁₀ = L, T, G; X₁₁ = K, R; X₁₂ = N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, Y; X₁₃ = P, A, C, D, E, F, G, H, I, K, L,  M, N, Q, R, S, T, V, W, Y; X₁₄ = V, I; X₁₅ = F, L, V; X₁₆ = F, V  46 D1 domain v10EEEX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVG PIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀T X₁₁RENMDFSISISX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGS  PDTEX₁₆KSGAGTELSVRAKPS —Amino acid X₁ = L, I, V; X₂ = V, L, I; X₃ = A, V;  substitutionsX₄ = V, I, L; X₅ = I, T, S, F;  relative toX₆ = E, V, L; X₇ = K, R; X₈ = E, Q;  SEQ ID NO: 46X₉ = H, P, R; X₁₀ = S, T, G; X₁₁ = K, R; X₁₂ = N, A, C, D, E, F, G, H, I, K, L,  M, P, Q, R, S, T, V, W, Y; X₁₃ = P, A, C, D, E, F, G, H, I, K, L,  M, N, Q, R, S, T, V, W, Y; X₁₄ = V, I; X₁₅ = F, L, V; X₁₆ = F, V  47 Pan D1 domainEEX₁X₂QX₃IQPDKX₄VX₅VAAGEX₆X₇X₈LX₉CTX₁₀TS LX₁₁PVGPIQWFRGAGPX₁₂RX₁₃LIYNQX₁₄X₁₅GX₁₆FP RVTTVSX₁₇X₁₈TX₁₉RX₂₀NMDFX₂₁IX₂₂IX₂₃X₂₄ITX₂₅AD AGTYYCX₂₆KX₂₇RKGSPDX₂₈X₂₉EX₃₀KSGAGTELSVR  X₃₁KPS — Amino acidX₁ = E, G; X₂ = L, I, V; X₃ = V, L, I;  substitutionsX₄ = S, F; X₅ = L, S; X₆ = S, T; X₇ = A, V;  relative toX₈ = I, T; X₉ = H, R, L; X₁₀ = A, V, I, L;  SEQ ID NO: 47X₁₁ = I, T, S, F; X₁₂ = A, G; X₁₃ = E, V, L; X₁₄ = K, R; X₁₅ = E, Q; X₁₆ = H, P, R; X₁₇ = D, E; X₁₈ = S, L, T, G; X₁₉ = K, R; X₂₀ = E, N; X₂₁ = S, P; X₂₂ = S, R;  X₂₃ = S, G; X₂₄ = any amino acid; X₂₅ = any amino acid; X₂₆ = V, I;  X₂₇ = F, L, V; X₂₈ = D or absent; X₂₉ = T, V; X₃₀ = F, V; and X₃₁ = A, G  48 Pan D1 domainEEELQX₁IQPDKSVX₂VAAGEX₃AX₄LX₅CTX₆TSLX₇P VGPIQWFRGAGPX₈RX₉LIYNQX₁₀X₁₁GX₁₂FPRVTTV SX₁₃X₁₄TKRX₁₅NMDFSIX₁₆IX₁₇X₁₈ITPADAGTYYCX₁₉ KFRKGX₂₀X₂₁X₂₂DX₂₃EFKSGAGTELSVRAKPS — Amino acidX₁ = V, I; X₂ = L, S; X₃ = T, S;  substitutionsX₄ = T, I; X₅ = R, H; X₆ = A, V, I;  relative toX₇ = I, R, Y, K, F; X₈ = G, A;  SEQ ID NO: 48X₉ = E, V; X₁₀ = K, R; X₁₁ = E, D, Q; X₁₂ = H, P; X₁₃ = D, E; X₁₄ = S, L, T; X₁₅ = N, E; X₁₆ = R, S; X₁₇ = G, S;  X₁₈ = N, A; X₁₉ = V, I; X₂₀ = S, I, M; X₂₁ = P or absent;  X₂₂ = D, P; and X₂₃ = V, T  49Pan D1 domain EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGP IQWFRGAGPGRX₄LIYNQX₅X₆GX₇FPRVTTVSDX₈TK RNNMDFSIRIGX₉ITPADAGTYYCX₁₀KFRKGSPDDV  EFKSGAGTELSVRAKPS — Amino acidX₁ = V, I, L; X₂ = A, I, V, L;  substitutionsX₃ = I, F, S, T; X₄ = E, V, L;  relative toX₅ = K, R; X₆ = E, Q; X₇ = H, P, R;  SEQ ID NO: 49X₈ = L, T, S, G; X₉ =  A; and  X₁₀ = V, I  50 Pan D1 domainEEELQX₁IQPDKSVSVAAGESAILHCTX₂TSLX₃PVGPI QWFRGAGPARX₄LIYNQX₅X₆GX₇FPRVTTVSEX₈TK RENMDFSISISX₉ITPADAGTYYCX₁₀KFRKGSPDTEF  KSGAGTELSVRAKPS — Amino acidX₁ = V, I; X₂ = V, I; X₃ = I, F;  substitutionsX₄ = E, V; X₅ = K, R; X₆ = E, Q;  relative toX₇ = H, P; X₈ = S, T; X₉ = N, A;  SEQ ID NO: 50 and X₁₀ = V, I  51Pan D1 domain EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGP IQWFRGAGPGRX₄LIYNQX₅EGX₆FPRVTTVSDX₇TK RNNMDFSIRIGX₈ITPADAGTYYCX₉KFRKGSPDDVE  FKSGAGTELSVRAKPS — Amino acidX₁ = V, I; X₂ = A, I; X₃ = I, F;  substitutionsX₄ = E, V; X₅ = K, R; X₆ = H, P;  relative to X₇ = L, T; X₈ = N, A; and SEQ ID NO: 51 X₉ = V, I  52 Pan D1 domainEEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGP IQWFRGAGPGRELIYNQX₄EGX₅FPRVTTVSDX₆TKR NNMDFSIRIGX₇ITPADAGTYYCVKFRKGSPDDVEF  KSGAGTELSVRAKPS — Amino acidX₁ = V, L, I; X₂ = A, I, L;  substitutions X₃ = I, T, S, F; X₄ = K, R; relative to X₅ = H, P, R; X₆ = L, T, G;  SEQ ID NO: 52 and X₇ = N, A 212Pan D1 domain EEELQX₁IQPDKSVSVAAGESAILHCTX₂TSLX₃PVGPI QWFRGAGPARELIYNQX₄EGX₅FPRVTTVSEX₆TKRE NMDFSISISX₇ITPADAGTYYCVKFRKGSPDTEFKSG  AGTELSVRAKPS — Amino acidX₁ = V, L, I; X₂ = V, I, L;  substitutions X₃ = I, T, S, F; X₄ = K, R; relative to X₅ = H, P, R; X₆ = S, T, G;  SEQ ID NO: 212 and X₇ = N, A217 Pan D1 domain EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGP IQWFRGAGPGRX₄LIYNQX₅X₆GX₇FPRVTTVSDX₈TK RNNMDFSIRIGX₉X₁₀X₁₁X₁₂ADAGTYYCX₁₃KFRKGSP  DDVEFKSGAGTELSVRAKPS —Amino acid X₁ = V, L, or I; X₂ = A, V, L, or I;  substitutionsX₃ = I, S, T, or F; X₄ = E, L, or V;  relative toX₅ = K or R; X₆ = E or Q; X₇ = H, R or P;  SEQ ID NO: 217X₈ = S, G, L or T, X₉ = any amino acid; X₁₀ = any amino acid; X₁₁ = any amino acid; X₁₂ = any amino acid; and X₁₃ = V or I 218 Pan D1 domainEEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGP IQWFRGAGPGRX₄LIYNQX₅X₆GX₇FPRVTTVSDX₈TK RNNMDFSIRIGX₉ITX₁₀ADAGTYYCX₁₁KFRKGSPDD  VEFKSGAGTELSVRAKPS — Amino acidX₁ = V, L or I; X₂ = A, V, L, or I;  substitutionsX₃ = I, S, T or F; X₄ = E, L, or V;  relative toX₅ = K or R; X₆ = E or Q; X₇ = H, R or P;  SEQ ID NO: 218X₈ = S, G, L, or T; X₉ = N;  X₁₀ = any amino acid other than P; and X₁₁ = V or I

In some embodiments, a polypeptide includes a SIRP-α D1 variant having asequence of:EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRIGX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDDVEX₁₆KSGAGTELSVRAKPS (SEQ ID NO: 37), wherein X₁ is L, I, or V; X₂ is V, L, or, I;X₃ is A or V; X₄ is A, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L;X₇ is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is L, T, or G; X₁₁ isK or R; X₁₂ is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W,or Y; X₁₃ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, orY; X₁₄ is V or I; X₁₅ is F, L, or V; and X₁₆ is F or V; and wherein thevariant has at least one amino acid substitution relative to a wild-typeSIRP-α D1 domain having the sequence of SEQ ID NO: 1.

In some embodiments, a polypeptide includes a SIRP-α D1 variant having asequence of:EEGX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRIGX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDDVEX₁₆KSGAGTELSVRAKPS (SEQ ID NO: 40), wherein X₁ is L, I, or V; X₂ is V, L, or, I;X₃ is A or V; X₄ is A, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L;X₇ is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is L, T, or G; X₁₁ isK or R; X₁₂ is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W,or Y; X₁₃ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, orY; X₁₄ is V or I; X₁₅ is F, L, or V; and X₁₆ is F or V; and wherein thevariant has at least one amino acid substitution relative to a wild-typeSIRP-α D1 domain having the sequence of SEQ ID NO: 4.

In some embodiments, a polypeptide includes a SIRP-α D1 variant having asequence of:EEEX₁QX₂IQPDKFVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRIGX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDDVEX₁₆KSGAGTELSVRAKPS (SEQ ID NO: 41), wherein X₁ is L, I, or V; X₂ is V, L, or, I;X₃ is A or V; X₄ is A, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L;X₇ is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is L, T, or G; X₁₁ isK or R; X₁₂ is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W,or Y; X₁₃ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, orY; X₁₄ is V or I; X₁₅ is F, L, or V; and X₁₆ is F or V; and wherein thevariant has at least one amino acid substitution relative to a wild-typeSIRP-α D1 domain having the sequence of SEQ ID NO: 5.

In some embodiments, a polypeptide includes a SIRP-α D1 variant having asequence of:EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFPIRIGX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDDVEX₁₆KSGAGTELSVRAKPS (SEQ ID NO: 42), and wherein X₁ is L, I, or V; X₂ is V, L, or,I; X₃ is A or V; X₄ is A, I, or L; X₅ is I, T, S, or F; X₆ is E, V, orL; X₇ is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is L, T, or G; X₁₁is K or R; X₁₂ is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V,W, or Y; X₁₃ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W,or Y; X₁₄ is V or I; X₁₅ is F, L, or V; and X₁₆ is F or V; and whereinthe variant has at least one amino acid substitution relative to awild-type SIRP-α D1 domain having the sequence of SEQ ID NO: 6.

In some embodiments, a polypeptide includes a SIRP-α D1 variant having asequence of:EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPGRX₆LIYNQX₇X₈GX₉FPRVTTVSDX₁₀TX₁₁RNNMDFSIRISX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDDVEX₁₆KSGAGTELSVRAKPS (SEQ ID NO: 45), and wherein X₁ is L, I, or V; X₂ is V, L, or,I; X₃ is A or V; X₄ is A, I, or L; X₅ is I, T, S, or F; X₆ is E, V, orL; X₇ is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is L, T, or G; X₁₁is K or R; X₁₂ is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V,W, or Y; X₁₃ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W,or Y; X₁₄ is V or I; X₁₅ is F, L, or V; and X₁₆ is F or V; and whereinthe variant has at least one amino acid substitution relative to awild-type SIRP-α D1 domain having the sequence of SEQ ID NO: 9.

In any of the aforementioned embodiments in this aspect of thedisclosure, a polypeptide includes a SIRP-α D1 variant having a sequenceof any one of SEQ ID NOs: 37, 40-42, and 45, wherein X₁ is L, I, or V.In any of the aforementioned embodiments, X₂ is V, L, or, I. In any ofthe aforementioned embodiments, X₃ is A or V. In any of theaforementioned embodiments, X₄ is A, I, or L. In any of theaforementioned embodiments, X₅ is I, T, S, or F. In any of theaforementioned embodiments, X₆ is E, V, or L. In any of theaforementioned embodiments, X₇ is K or R. In any of the aforementionedembodiments, X₈ is E or Q. In any of the aforementioned embodiments, X₉is H, P, or R. In any of the aforementioned embodiments, X₁₀ is L, T, orG. In any of the aforementioned embodiments, X₁₁ is K or R. In any ofthe aforementioned embodiments, X₁₂ is N, A, C, D, E, F, G, H, I, K, L,M, P, Q, R, S, T, V, W, or Y. In any of the aforementioned embodiments,X₁₃ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y. Inany of the aforementioned embodiments, X₁₄ is V or I. In any of theaforementioned embodiments, X₁₅ is F, L, V. In any of the aforementionedembodiments, X₁₆ is F or V.

In some embodiments, a polypeptide provided herein includes no more thanten amino acid substitutions relative to the wild-type SIRP-α D1 domainhaving the sequence of any one of SEQ ID NOs: 1, 4-6, and 9. In someembodiments, the polypeptide provided herein includes no more than sevenamino acid substitutions relative to the wild-type SIRP-α D1 domainhaving the sequence of any one of SEQ ID NOs: 1, 4-6, and 9.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRP-α D1 domain having thesequence of any one of SEQ ID NOs: 1, 4-6, and 9. In some embodiments,the polypeptide binds CD47 with at least 100-fold greater bindingaffinity than the wild-type SIRP-α D1 domain having the sequence of anyone of SEQ ID NOs: 1, 4-6, and 9. In some embodiments, the polypeptidebinds CD47 with at least 1000-fold greater binding affinity than thewild-type SIRP-α D1 domain having the sequence of any one of SEQ ID NOs:1, 4-6, and 9. In some embodiments, a SIRP-α D1 variant polypeptide orfragment thereof binds to CD47 with a K_(D) less than 1×10⁻⁸ M, lessthan 5×10⁻⁹ M, less than 1×10⁻⁹ M, less 5×10⁻¹⁰ M, less than 1×10⁻¹⁰ Mor less than 1×10^(−11M). In some embodiments, a SIRP-α D1 variantpolypeptide or fragment thereof binds to CD47 with a K_(D) between about500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nMand 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM,between about 1 nM and 500 pM, between about 500 pM and 100 pM, betweenabout 100 pM and 50 pM, or between about 50 pM and 10 pM.

In some embodiments, a polypeptide includes a SIRP-α D1 variant having asequence of:EEEX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDTEX₁₆KSGAGTELSVRAKPS (SEQ ID NO: 38), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃is A or V; X₄ is V, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is S, T, or G; X₁₁ is Kor R; X₁₂ is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, orY; X₁₃ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y;X₁₄ is V or I; X₁₅ is F, L, or V; and X₁₆ is F or V; and wherein thevariant has at least one amino acid substitution relative to a wild-typeSIRP-α D1 domain having the sequence of SEQ ID NO: 2.

In some embodiments, a polypeptide includes a SIRP-α D1 variant having asequence of:EEEX₁QX₂IQPDKSVSVAAGESX₃ILLCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDTEX₁₆KSGAGTELSVRAKPS (SEQ ID NO: 39), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃is A or V; X₄ is V, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is S, T, or G; X₁₁ is Kor R; X₁₂ is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, orY; X₁₃ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y;X₁₄ is V or I; X₁₅ is F, L, or V; and X₁₆ is F or V; and wherein thevariant has at least one amino acid substitution relative to a wild-typeSIRP-α D1 domain having the sequence of SEQ ID NO: 3.

In some embodiments, a polypeptide includes a SIRP-α D1 variant having asequence of:EEEX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDTEX₁₆KSGAGTELSVRGKPS (SEQ ID NO: 43), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃is A or V; X₄ is V, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is S, T, or G; X₁₁ is Kor R; X₁₂ is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, orY; X₁₃ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y;X₁₄ is V or I; X₁₅ is F, L, or V; and X₁₆ is F or V; and wherein thevariant has at least one amino acid substitution relative to a wild-typeSIRP-α D1 domain having the sequence of SEQ ID NO: 7.

In some embodiments, a polypeptide includes a SIRP-α D1 variant having asequence of:EEEX₁QX₂IQPDKSVSVAAGESX₃ILHCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDTEX₁₆KSGAGTELSVRAKPS (SEQ ID NO: 46), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃is A or V; X₄ is V, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is S, T, or G; X₁₁ is Kor R; X₁₂ is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, orY; X₁₃ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y;X₁₄ is V or I; X₁₅ is F, L, or V; and X₁₆ is F or V; and wherein thevariant has at least one amino acid substitution relative to a wild-typeSIRP-α D1 domain having the sequence of SEQ ID NO: 10.

In any of the aforementioned embodiments in this aspect of thedisclosure, a polypeptide includes a SIRP-α D1 variant having a sequenceof any one of SEQ ID NOs: 38, 39, 43, and 46, wherein X₁ is L, I, or V.In any of the aforementioned embodiments, X₂ is V, L, or, I. In any ofthe aforementioned embodiments, X₃ is A or V. In any of theaforementioned embodiments, X₄ is V, I, or L. In any of theaforementioned embodiments, X₅ is I, T, S, or F. In any of theaforementioned embodiments, X₆ is E, V, or L. In any of theaforementioned embodiments, X₇ is K or R. In any of the aforementionedembodiments, X₈ is E or Q. In any of the aforementioned embodiments, X₉is H, P, or R. In any of the aforementioned embodiments, X₁₀ is S, T, orG. In any of the aforementioned embodiments, X₁₁ is K or R. In any ofthe aforementioned embodiments, X₁₂ is N, A, C, D, E, F, G, H, I, K, L,M, P, Q, R, S, T, V, W, or Y. In any of the aforementioned embodiments,X₁₃ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y. Inany of the aforementioned embodiments, X₁₄ is V or I. In any of theaforementioned embodiments, X₁₅ is F, L, or V. In any of theaforementioned embodiments, X₁₆ is F or V.

In some embodiments, a polypeptide includes a SIRP-α D1 variant havingno more than ten amino acid substitutions relative to the wild-typeSIRP-α D1 domain having the sequence of any one of SEQ ID NOs: 2, 3, 7,and 10. In some embodiments, a polypeptide includes a SIRP-α D1 varianthaving no more than seven amino acid substitutions relative to thewild-type SIRP-α D1 domain having the sequence of any one of SEQ ID NOs:2, 3, 7, and 10.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRP-α D1 domain having thesequence of any one of SEQ ID NOs: 2, 3, 7, and 10. In some embodiments,the polypeptide binds CD47 with at least 100-fold greater bindingaffinity than the wild-type SIRP-α D1 domain having the sequence of anyone of SEQ ID NOs: 2, 3, 7, and 10. In some embodiments, the polypeptidebinds CD47 with at least 1000-fold greater binding affinity than thewild-type SIRP-α D1 domain having the sequence of any one of SEQ ID NOs:2, 3, 7, and 10. In some embodiments, a SIRP-α D1 variant polypeptide orfragment thereof binds to CD47 with a K_(D) less than 1×10⁻⁸ M, lessthan 5×10⁻⁹ M, less than 1×10⁻⁹ M, less 5×10⁻¹⁰ M, less than 1×10⁻¹⁰ Mor less than 1×10⁻¹¹ M. In some embodiments, a SIRP-α D1 variantpolypeptide or fragment thereof binds to CD47 with a K_(D) between about500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nMand 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM,between about 1 nM and 500 pM, between about 500 pM and 100 pM, betweenabout 100 pM and 50 pM, or between about 50 pM and 10 pM.

In some embodiments, a polypeptide includes a SIRP-α D1 variant having asequence of:EEEX₁QX₂IQPDKSVLVAAGETX₃TLRCTX₄TSLX₅PVGPIQWFRGAGPARX₆LIYNQX₇X₈GX₉FPRVTTVSEX₁₀TX₁₁RENMDFSISISX₁₂ITX₁₃ADAGTYYCX₁₄KX₁₅RKGSPDTEX₁₆KSGAGTELSVRAKPS (SEQ ID NO: 44), wherein X₁ is L, I, or V; X₂ is V, L, or, I; X₃is A or V; X₄ is A, I, or L; X₅ is I, T, S, or F; X₆ is E, V, or L; X₇is K or R; X₈ is E or Q; X₉ is H, P, or R; X₁₀ is S, T, or G; X₁₁ is Kor R; X₁₂ is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, orY; X₁₃ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y;X₁₄ is V or I; X₁₅ is F, L, or V; and X₁₆ is F or V; and wherein thevariant has at least one amino acid substitution relative to a wild-typeSIRP-α D1 domain having the sequence of SEQ ID NO: 8.

In some embodiments, the polypeptide has the sequence of SEQ ID NO: 44,wherein X₁ is L, I, or V. In any of the aforementioned embodiments inthis aspect of the disclosure, X₂ is V, L, or, I. In any of theaforementioned embodiments, X₃ is A or V. In any of the aforementionedembodiments, X₄ is A, I, or L. In any of the aforementioned embodiments,X₅ is I, T, S, or F. In any of the aforementioned embodiments, X₆ is E,V, or L. In any of the aforementioned embodiments, X₇ is K or R. In anyof the aforementioned embodiments, X₈ is E or Q. In any of theaforementioned embodiments, X₉ is H, P, or R. In any of theaforementioned embodiments, X₁₀ is S, T, or G. In any of theaforementioned embodiments, X₁₁ is K or R. In any of the aforementionedembodiments, X₁₂ is N, A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T,V, W, or Y. In any of the aforementioned embodiments, X₁₃ is P, A, C, D,E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y. In any of theaforementioned embodiments, X₁₄ is V or I. In any of the aforementionedembodiments, X₁₅ is F, L, or V. In any of the aforementionedembodiments, X₁₆ is F or V.

In some embodiments, a polypeptide includes a SIRP-α D1 variant havingno more than ten amino acid substitutions relative to the wild-typeSIRP-α D1 domain having the sequence of SEQ ID NO: 8. In someembodiments, a polypeptide includes a SIRP-α D1 variant having no morethan seven amino acid substitutions relative to the wild-type SIRP-α D1domain having the sequence of SEQ ID NO: 8.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRP-α D1 domain having thesequence of SEQ ID NO: 8. In some embodiments, the polypeptide bindsCD47 with at least 100-fold greater binding affinity than the wild-typeSIRP-α D1 domain having the sequence of SEQ ID NO: 8. In someembodiments, the polypeptide binds CD47 with at least 1000-fold greaterbinding affinity than the wild-type SIRP-α D1 domain having the sequenceof SEQ ID NO: 8. In some embodiments, a SIRP-α D1 variant polypeptide orfragment thereof binds to CD47 with a K_(D) less than 1×10⁻⁸ M, lessthan 5×10⁻⁹ M, less than 1×10⁻⁹ M, less 5×10⁻¹⁰ M, less than 1×10⁻¹⁰ Mor less than 1×10⁻¹¹M. In some embodiments, a SIRP-α D1 variantpolypeptide or fragment thereof binds to CD47 with a K_(D) between about500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nMand 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM,between about 1 nM and 500 pM, between about 500 pM and 100 pM, betweenabout 100 pM and 50 pM, or between about 50 pM and 10 pM.

In another aspect, the disclosure features a polypeptide including aSIRP-α D1 variant having a sequence of:

EEX₁X₂QX₃IQPDKX₄VX₅VAAGEX₆X₇X₈LX₉CTX₁₀TSLX₁₁PVGPIQWFRGAGPX₁₂RX₁₃LIYNQX₁₄X₁₅GX₁₆FPRVTTVSX₁₇X₁₈TX₁₉RX₂₀NMDFX₂₁IX₂₂IX₂₃X₂₄ITX₂₅ADAGTYYCX₂₆KX₂₇RKGSPDX₂₈X₂₉EX₃₀KSGAGTELSVRX₃₁KPS (SEQ ID NO: 47), wherein X₁ is E or G; X₂is L, I, or V; X₃ is V, L, or, I; X₄ is S or F; X₅ is L or S; X₆ is S orT; X₇ is A or V; X₈ is I or T; X₉ is H or R; X₁₀ is A, V, I, or L; X₁₁is I, T, S, or F; X₁₂ is A or G; X₁₃ is E, V, or L; X₁₄ is K or R; X₁₅is E or Q; X₁₆ is H, P, or R; X₁₇ is D or E; X₁₈ is S, L, T, or G; X₁₉is K or R; X₂₀ is E or N; X₂₁ is S or P; X₂₂ is S or R; X₂₃ is S or G;X₂₄ is any amino acid; X₂₅ is any amino acid; X₂₆ is V or I; X₂₇ is F,L, V; X₂₈ is D or absent; X₂₉ is T or V; X₃₀ is F or V; and X₃₁ is A orG; and wherein the variant has at least one amino acid substitutionrelative to a wild-type SIRP-α D1 domain having the sequence of any oneof SEQ ID NOs: 1-10.

In some embodiments, the polypeptide has the sequence of SEQ ID NO: 47,wherein X₁ is E or G. In any of the aforementioned embodiments in thisaspect of the disclosure, X₂ is L, I, or V. In any of the aforementionedembodiments, X₃ is V, L, or, I. In any of the aforementionedembodiments, X₄ is S or F. In any of the aforementioned embodiments, X₅is L or S. In any of the aforementioned embodiments, X₆ is S or T. Inany of the aforementioned embodiments, X₇ is A or V. In any of theaforementioned embodiments, X₈ is I or T. In any of the aforementionedembodiments, X₉ is H or R. In any of the aforementioned embodiments, X₁₀is A, V, I, or L. In any of the aforementioned embodiments, X₁₁ is I, T,S, or F. In any of the aforementioned embodiments, X₁₂ is A or G. In anyof the aforementioned embodiments, X₁₃ is E, V, or L. In any of theaforementioned embodiments, X₁₄ is K or R. In any of the aforementionedembodiments, X₁₅ is E or Q. In any of the aforementioned embodiments,X₁₆ is H, P, or R. In any of the aforementioned embodiments, X₁₇ is D orE. In any of the aforementioned embodiments, X₁₈ is S, L, T, or G. Inany of the aforementioned embodiments, X₁₉ is K or R. In any of theaforementioned embodiments, X₂₀ is E or N. In any of the aforementionedembodiments, X₂₁ is S or P. In any of the aforementioned embodiments,X₂₂ is S or R. In any of the aforementioned embodiments, X₂₃ is S or G.In any of the aforementioned embodiments, X₂₄ is N, A, C, D, E, F, G, H,I, K, L, M, P, Q, R, S, T, V, W, or Y. In any of the aforementionedembodiments, X₂₅ is P, A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T,V, W, or Y. In any of the aforementioned embodiments, X₂₆ is V or I. Inany of the aforementioned embodiments, X₂₇ is F, L, V. In any of theaforementioned embodiments, X₂₈ is D or absent. In any of theaforementioned embodiments, X₂₉ is T or V. In any of the aforementionedembodiments, X₃₀ is F or V. In any of the aforementioned embodiments,X₃₁ is A or G.

In some embodiments, the polypeptide of this aspect of the disclosureincludes no more than ten amino acid substitutions relative to thewild-type SIRP-α D1 domain having the sequence of any one of SEQ ID NOs:1-10. In some embodiments, the polypeptide of this aspect of thedisclosure includes no more than seven amino acid substitutions relativeto the wild-type SIRP-α D1 domain having the sequence of any one of SEQID NOs: 1-10.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRP-α D1 domain having thesequence of any one of SEQ ID NOs: 1-10. In some embodiments, thepolypeptide binds CD47 with at least 100-fold greater binding affinitythan the wild-type SIRP-α D1 domain having the sequence of any one ofSEQ ID NOs: 1-10. In some embodiments, the polypeptide binds CD47 withat least 1000-fold greater binding affinity than the wild-type SIRP-α D1domain having the sequence of any one of SEQ ID NOs: 1-10. In someembodiments, a SIRP-α D1 variant polypeptide or fragment thereof bindsto CD47 with a K_(D) less than 1×10⁻⁸ M, less than 5×10⁻⁹ M, less than1×10⁻⁹ M, less 5×10⁻¹⁰ M, less than 1×10⁻¹⁰ M or less than 1×10⁻¹¹ M. Insome embodiments, a SIRP-α D1 variant polypeptide or fragment thereofbinds to CD47 with a K_(D) between about 500 nM and 100 nM, betweenabout 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM,between about 500 pM and 100 pM, between about 100 pM and 50 pM, orbetween about 50 pM and 10 pM.

In some embodiments, a polypeptide includes a SIRP-α D1 variant having asequence of:

EEELQX₁IQPDKSVX₂VAAGEX₃AX₄LX₅CTX₆TSLX₇PVGPIQWFRGAGPX₈RX₉LIYNQX₁₀X₁₁GX₁₂FPRVTTVSX₁₃X₁₄TKRX₁₅NMDFSIX₁₆IX₁₇X₁₈ITPADAGTYYCX₁₉KFRKGX₂₀X₂₁X₂₂DX₂₃EFKSGAGTELSVRAKPS (SEQ ID NO: 48), wherein X₁ is V or I; X₂ is Lor S; X₃ is T or S; X₄ is T or I; X₅ is R or H; X₆ is A, V, or I; X₇ isI, R, Y, K or F; X₈ is G or A; X₉ is E or V; X₁₀ is K or R; X₁₁ is E, Dor Q; X₁₂ is H or P; X₁₃ is D or E; X₁₄ is S, L or T; X₁₅ is N or E; X₁₆is R or S; X₁₇ is G or S; X₁₈ is N or A; X₁₉ is V or I; X₂₀ is S, I orM; X₂₁ is P or absent; X₂₂ is D or P; and X₂₃ is V or T, or a fragmentthereof.

In another aspect, the disclosure features a polypeptide including aSIRP-α D1 variant having a sequence of:

EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅X₆GX₇FPRVTTVSDX₈TKRNNMDFSIRIGX₉ITPADAGTYYCX₁₀KFRKGSPDDVEFKSGAGTELSVRAK PS (SEQID NO: 49), wherein X₁ is V, L, or I; X₂ is A, I, V, or L; X₃ is I, F,S, or T; X₄ is E, V, or L; X₅ is K or R; X₆ is E or Q; X₇ is H, P, or R;X₈ is L, T, S, or G; X₉ is A; and X₁₀ is V or I; and wherein the varianthas at least one amino acid substitution relative to a wild-type SIRP-αD1 domain having the sequence of any one of SEQ ID NO: 1.

In some embodiments, the polypeptide has the sequence of SEQ ID NO: 49,wherein X₁ is V, L or I. In any of the aforementioned embodiments inthis aspect of the disclosure, X₂ is A, I, V, or L. In any of theaforementioned embodiments, X₃ is I, F, S, or T. In any of theaforementioned embodiments, X₄ is E, V, or L. In any of theaforementioned embodiments, X₅ is K or R. In any of the aforementionedembodiments, X₆ is E or Q. In any of the aforementioned embodiments, X₇is H, P, or R. In any of the aforementioned embodiments, X₈ is L, T, Sor G. In any of the aforementioned embodiments, X₉ is A. In any of theaforementioned embodiments, X₁₀ is V or I.

In some embodiments, the polypeptide has a high affinity SIRP-α D1domain having at least 85% sequence identity (e.g., at least 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity) to SEQ ID NO: 49, wherein each of X₁, X₂, X₃, X₄, X₅,X₆, X₇, X₈, X₉, and X₁₀ are not a wild-type amino acid.

In some embodiments, the polypeptide of this aspect of the disclosureincludes no more than ten amino acid substitutions relative to thewild-type SIRP-α D1 domain having the sequence of any one of SEQ IDNO: 1. In some embodiments, the polypeptide of this aspect of thedisclosure includes no more than seven amino acid substitutions relativeto the wild-type SIRP-α D1 domain having the sequence of any one of SEQID NO: 1.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRP-α D1 domain having thesequence of any one of SEQ ID NO: 1. In some embodiments, thepolypeptide binds CD47 with at least 100-fold greater binding affinitythan the wild-type SIRP-α D1 domain having the sequence of any one ofSEQ ID NO: 1. In some embodiments, the polypeptide binds CD47 with atleast 1000-fold greater binding affinity than the wild-type SIRP-α D1domain having the sequence of any one of SEQ ID NO: 1. In someembodiments, a SIRP-α D1 variant polypeptide or fragment thereof bindsto CD47 with a K_(D) less than 1×10⁻⁸ M, less than 5×10⁻⁹ M, less than1×10⁻⁹ M, less 5×10⁻¹⁰ M, less than 1×10⁻¹⁰ M or less than 1×10⁻¹¹ M. Insome embodiments, a SIRP-α D1 variant polypeptide or fragment thereofbinds to CD47 with a K_(D) between about 500 nM and 100 nM, betweenabout 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM,between about 500 pM and 100 pM, between about 100 pM and 50 pM, orbetween about 50 pM and 10 pM.

In another aspect, the disclosure features a polypeptide including aSIRP-α D1 variant having a sequence of:

EEELQX₁IQPDKSVSVAAGESAILHCTX₂TSLX₃PVGPIQWFRGAGPARX₄LIYNQX₅X₆GX₇FPRVTTVSEX₈TKRENMDFSISISX₉ITPADAGTYYCX₁₀KFRKGSPDTEFKSGAGTELSVRAKPS, (SEQ IDNO: 50), wherein X₁ is V or I; X₂ is V or I; X₃ is I or F; X₄ is E or V;X₅ is K or R; X₆ is E or Q; X₇ is H or P; X₈ is S or T; X₉ is N or A;and X₁₀ V or I; and wherein the variant has at least one amino acidsubstitution relative to a wild-type SIRP-α D1 domain having thesequence of any one of SEQ ID NO: 2.

In some embodiments, the polypeptide has the sequence of SEQ ID NO: 50,wherein X₁ is V or I. In any of the aforementioned embodiments in thisaspect of the disclosure, X₂ is V or I. In any of the aforementionedembodiments, X₃ is I or F. In any of the aforementioned embodiments, X₄is E or V. In any of the aforementioned embodiments, X₅ is K or R. Inany of the aforementioned embodiments, X₆ is E or Q. In any of theaforementioned embodiments, X₇ is H or P. In any of the aforementionedembodiments, X₈ is S or R. In any of the aforementioned embodiments, X₉is N or A. In any of the aforementioned embodiments, X₁₀ is V or I.

In some embodiments, the polypeptide has a high affinity SIRP-α D1domain having at least 85% sequence identity (e.g., at least 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity) to SEQ ID NO: 50, wherein each of X₁, X₂, X₃, X₄, X₅,X₆, X₇, X₈, X₉, and X₁₀ is not a wild-type amino acid.

In some embodiments, the polypeptide of this aspect of the disclosureincludes no more than ten amino acid substitutions relative to thewild-type SIRP-α D1 domain having the sequence of any one of SEQ ID NO:2. In some embodiments, the polypeptide of this aspect of the disclosureincludes no more than seven amino acid substitutions relative to thewild-type SIRP-α D1 domain having the sequence of any one of SEQ ID NO:2.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRP-α D1 domain having thesequence of any one of SEQ ID NO: 2. In some embodiments, thepolypeptide binds CD47 with at least 100-fold greater binding affinitythan the wild-type SIRP-α D1 domain having the sequence of any one ofSEQ ID NO: 2. In some embodiments, the polypeptide binds CD47 with atleast 1000-fold greater binding affinity than the wild-type SIRP-α D1domain having the sequence of any one of SEQ ID NO: 2. In someembodiments, a SIRP-α D1 variant polypeptide or fragment thereof bindsto CD47 with a K_(D) less than 1×10⁻⁸ M, less than 5×10⁻⁹ M, less than1×10⁻⁹ M, less 5×10⁻¹⁰ M, less than 1×10⁻¹⁰ M or less than 1×10⁻¹¹ M. Insome embodiments, a SIRP-α D1 variant polypeptide or fragment thereofbinds to CD47 with a K_(D) between about 500 nM and 100 nM, betweenabout 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM,between about 500 pM and 100 pM, between about 100 pM and 50 pM, orbetween about 50 pM and 10 pM.

In another aspect, the disclosure features a polypeptide including aSIRP-α D1 variant having a sequence of:

EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅EGX₆FPRVTTVSDX₇TKRNNMDFSIRIGX₈ITPADAGTYYCX₉KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ IDNO: 51), wherein X₁ is V or I; X₂ is A or I; X₃ is I or F; X₄ is E or V;X₅ is K or R; X₆ is H or P; X₇ is L or T; X₈ is N or A; and X₉ is V orI; and wherein the variant has at least one amino acid substitutionrelative to a wild-type SIRP-α D1 domain having the sequence of any oneof SEQ ID NO: 1.

In some embodiments, the polypeptide has the sequence of SEQ ID NO: 51,wherein X₁ is V or I. In any of the aforementioned embodiments in thisaspect of the disclosure, X₂ is A or I. In any of the aforementionedembodiments, X₃ is I or F. In any of the aforementioned embodiments, X₄is E or V. In any of the aforementioned embodiments, X₅ is K or R. Inany of the aforementioned embodiments, X₆ is H or P. In any of theaforementioned embodiments, X₇ is L or T. In any of the aforementionedembodiments, X₈ is N or A. In any of the aforementioned embodiments, X₉is V or I. In some embodiments, X₄ is not V.

In some embodiments, the polypeptide has the sequence of SEQ ID NO: 51,wherein X₈ is A. In any of the aforementioned embodiments in this aspectof the disclosure, X₈ is A and X₁ is V or I. In any of theaforementioned embodiments in this aspect of the disclosure, X₈ is A andX₂ is A or I. In any of the aforementioned embodiments, X₈ is A and X₃is I or F. In any of the aforementioned embodiments, X₈ is A and X₄ is Eor V. In some embodiments, X₄ is not V. In any of the aforementionedembodiments, X₈ is A and X₅ is K or R. In any of the aforementionedembodiments, X₈ is A and X₆ is H or P. In any of the aforementionedembodiments, X₈ is A and X₇ is A or V. In any of the aforementionedembodiments, X₈ is A and X₉ is V or I.

In some embodiments, the polypeptide has the sequence of SEQ ID NO: 51,wherein X₈ is A. In any of the aforementioned embodiments in this aspectof the disclosure, X₈ is A and X₁ is I. In any of the aforementionedembodiments in this aspect of the disclosure, X₈ is A and X₂ is I. Inany of the aforementioned embodiments, X₈ is A and X₃ is F. In any ofthe aforementioned embodiments, X₈ is A and X₄ is V. In any of theaforementioned embodiments, X₈ is A and X₅ is R. In any of theaforementioned embodiments, X₈ is A and X₆ is P. In any of theaforementioned embodiments, X₈ is A and X₇ is T. In any of theaforementioned embodiments, X₈ is A and X₉ is I.

In some embodiments, the polypeptide has a high affinity SIRP-α D1domain having at least 85% sequence identity (e.g., at least 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity) to SEQ ID NO: 51, wherein each of X₁, X₂, X₃, X₄, X₅,X₆, X₇, X₈, and X₉ is not a wild-type amino acid.

In some embodiments, the polypeptide of this aspect of the disclosureincludes no more than ten amino acid substitutions relative to thewild-type SIRP-α D1 domain having the sequence of any one of SEQ IDNO: 1. In some embodiments, the polypeptide of this aspect of thedisclosure includes no more than seven amino acid substitutions relativeto the wild-type SIRP-α D1 domain having the sequence of any one of SEQID NO: 1.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRP-α D1 domain having thesequence of any one of SEQ ID NO: 1. In some embodiments, thepolypeptide binds CD47 with at least 100-fold greater binding affinitythan the wild-type SIRP-α D1 domain having the sequence of any one ofSEQ ID NOs: 1. In some embodiments, the polypeptide binds CD47 with atleast 1000-fold greater binding affinity than the wild-type SIRP-α D1domain having the sequence of any one of SEQ ID NO: 1. In someembodiments, a SIRP-α D1 variant polypeptide or fragment thereof bindsto CD47 with a K_(D) less than 1×10⁻⁸ M, less than 5×10⁻⁹ M, less than1×10⁻⁹ M, less 5×10⁻¹⁰ M, less than 1×10⁻¹⁰ M or less than 1×10⁻¹¹ M. Insome embodiments, a SIRP-α D1 variant polypeptide or fragment thereofbinds to CD47 with a K_(D) between about 500 nM and 100 nM, betweenabout 100 nM and 50 nM, between about 50 nM and 10 nM, between about 10nM and 5 nM, between about 5 nM and 1 nM, between about 1 nM and 500 pM,between about 500 pM and 100 pM, between about 100 pM and 50 pM, orbetween about 50 pM and 10 pM.

In another aspect, the disclosure features a polypeptide including aSIRP-α D1 variant having a sequence of:

EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRELIYNQX₄EGX₅FPRVTTVSDX₆TKRNNMDFSIRIGX₇ITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ IDNO: 52), wherein X₁ is V, L, or I; X₂ is A, I, or L; X₃ is I, T, S, orF; X₄ is K or R; X₅ is H or P; X₆ is L, T, of G; X₇ is N or A; andwherein the variant has at least one amino acid substitution relative toa wild-type SIRP-α D1 domain having a sequence according to SEQ ID NO:1.

In some embodiments, the polypeptide has the sequence of SEQ ID NO: 52,wherein X₁ is V, L, or I. In any of the aforementioned embodiments inthis aspect of the disclosure, X₂ is A, I, or L. In any of theaforementioned embodiments, X₃ is I, T, S, or F. In any of theaforementioned embodiments, X₄ is K or R. In any of the aforementionedembodiments, X₅ is H or P. In any of the aforementioned embodiments, X₆is L, T, or G. In any of the aforementioned embodiments, X₇ is N or A.

In some embodiments, the polypeptide has the sequence of SEQ ID NO: 52,wherein X₁ is V or I. In any of the aforementioned embodiments in thisaspect of the disclosure, X₂ is A or I. In any of the aforementionedembodiments, X₃ is I or F. In any of the aforementioned embodiments, X₄is K or R. In any of the aforementioned embodiments, X₅ is H or P. Inany of the aforementioned embodiments, X₆ is L or T. In any of theaforementioned embodiments, X₇ is N or A.

In some embodiments, the polypeptide has the sequence of SEQ ID NO: 52,wherein X₇ is A. In any of the aforementioned embodiments in this aspectof the disclosure, X₇ is A and X₁ is V or I. In any of theaforementioned embodiments in this aspect of the disclosure, X₇ is A andX₂ is A or I. In any of the aforementioned embodiments, X₇ is A and X₃is I or F. In any of the aforementioned embodiments, X₇ is A and X₄ is Kor R. In any of the aforementioned embodiments, X₇ is A and X₅ is H orP. In any of the aforementioned embodiments, X₇ is A and X₆ is L or T.

In some embodiments, the polypeptide has the sequence of SEQ ID NO: 52,wherein X₇ is A. In any of the aforementioned embodiments in this aspectof the disclosure, X₇ is A and X₁ is I. In any of the aforementionedembodiments in this aspect of the disclosure, X₇ is A and X₂ is I. Inany of the aforementioned embodiments, X₇ is A and X₃ is F. In any ofthe aforementioned embodiments, X₇ is A and X₄ is R. In any of theaforementioned embodiments, X₇ is A and X₅ is P. In any of theaforementioned embodiments, X₇ is A and X₆ is T.

In some embodiments, the polypeptide has a high affinity SIRP-α D1domain having at least 85% sequence identity (e.g., at least 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity) to SEQ ID NO: 52, wherein each of X₁, X₂, X₃, X₄, X₅,X₆, and X₇ is not a wild-type amino acid.

In some embodiments, the polypeptide of this aspect of the disclosureincludes no more than ten amino acid substitutions relative to thewild-type SIRP-α D1 domain having the sequence of any one of SEQ IDNO: 1. In some embodiments, the polypeptide of this aspect of thedisclosure includes no more than seven amino acid substitutions relativeto the wild-type SIRP-α D1 domain having the sequence of any one of SEQID NO: 1.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRP-α D1 domain having thesequence of any one of SEQ ID NO: 1. In some embodiments, thepolypeptide binds CD47 with at least 100-fold greater binding affinitythan the wild-type SIRP-α D1 domain having the sequence of any one ofSEQ ID NO: 1. In some embodiments, the polypeptide binds CD47 with atleast 1000-fold greater binding affinity than the wild-type SIRP-α D1domain having the sequence of any one of SEQ ID NO: 1. In someembodiments, fragments include polypeptides of less than 10 amino acidsin length, about 10 amino acids in length, about 20 amino acids inlength, about 30 amino acids in length, about 40 amino acids in length,about 50 amino acids in length, about 60 amino acids in length, about 70amino acids in length, about 80 amino acids in length, about 90 aminoacids in length, about 100 amino acids in length, or more than about 100amino acids in length. Fragments retain the ability to bind to CD47.Preferably, SIRP-α D1 variant polypeptides and fragments thereof bind toCD47 with a higher affinity than a SIRP-α polypeptide binds to CD47. Forexample, in some embodiments, a SIRP-α D1 variant polypeptide orfragment thereof binds to CD47 with a K_(D) less than 1×10⁻⁸ M, lessthan 5×10⁻⁹ M, less than 1×10⁻⁹ M, less 5×10⁻¹⁰ M, less than 1×10⁻¹⁰ Mor less than 1×10⁻¹¹M. In some embodiments, a SIRP-α D1 variantpolypeptide or fragment thereof binds to CD47 with a K_(D) between about500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nMand 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM,between about 1 nM and 500 pM, between about 500 pM and 100 pM, betweenabout 100 pM and 50 pM, or between about 50 pM and 10 pM.

In another aspect, the disclosure features a polypeptide including aSIRP-α D1 variant having a sequence of:

EEELQX₁IQPDKSVSVAAGESAILHCTX₂TSLX₃PVGPIQWFRGAGPARELIYNQX₄EGX₅FPRVTTVSEX₆TKRENMDFSISISX₇ITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS (SEQ ID NO:212), wherein X₁ is V, L, or I; X₂ is V, I, or L; X₃ is I, T, S, or F;X₄ is K or R; X₅ is H, P, or R; X₆ is S, T, of G; X₇ is N or A; andwherein the variant has at least one amino acid substitution relative toa wild-type SIRP-α D1 domain having the sequence of any one of SEQ IDNO: 2.

In some embodiments, the polypeptide has the sequence of SEQ ID NO: 212,wherein X₁ is V, L, or I. In any of the aforementioned embodiments inthis aspect of the disclosure, X₂ is V, I, or L. In any of theaforementioned embodiments, X₃ is I, T, S, or F. In any of theaforementioned embodiments, X₄ is K or R. In any of the aforementionedembodiments, X₅ is H or P. In any of the aforementioned embodiments, X₆is S, T, or G. In any of the aforementioned embodiments, X₇ is N or A.

In some embodiments, the polypeptide has the sequence of SEQ ID NO: 212,wherein X₁ is V or I. In any of the aforementioned embodiments in thisaspect of the disclosure, X₂ is V or I. In any of the aforementionedembodiments, X₃ is I or F. In any of the aforementioned embodiments, X₄is K or R. In any of the aforementioned embodiments, X₅ is H or P. Inany of the aforementioned embodiments, X₆ is S or T. In any of theaforementioned embodiments, X₇ is N or A.

In some embodiments, the polypeptide has the sequence of SEQ ID NO: 212,wherein X₇ is A. In any of the aforementioned embodiments in this aspectof the disclosure, X₇ is A and X₁ is V or I. In any of theaforementioned embodiments in this aspect of the disclosure, X₇ is A andX₂ is V or I. In any of the aforementioned embodiments, X₇ is A and X₃is I or F. In any of the aforementioned embodiments, X₇ is A and X₄ is Kor R. In any of the aforementioned embodiments, X₇ is A and X₅ is H orP. In any of the aforementioned embodiments, X₇ is A and X₆ is S or T.

In some embodiments, the polypeptide has the sequence of SEQ ID NO: 212,wherein X₇ is A. In any of the aforementioned embodiments in this aspectof the disclosure, X₇ is A and X₁ is I. In any of the aforementionedembodiments in this aspect of the disclosure, X₇ is A and X₂ is I. Inany of the aforementioned embodiments, X₇ is A and X₃ is F. In any ofthe aforementioned embodiments, X₇ is A and X₄ is R. In any of theaforementioned embodiments, X₇ is A and X₅ is P. In any of theaforementioned embodiments, X₇ is A and X₆ is T.

In some embodiments, the polypeptide has a high affinity SIRP-α D1domain having at least 85% sequence identity (e.g., at least 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity) to SEQ ID NO: 212, wherein each of X₁, X₂, X₃, X₄,X₅, X₆, and X₇ is not a wild-type amino acid.

In some embodiments, the polypeptide of this aspect of the disclosureincludes no more than ten amino acid substitutions relative to thewild-type SIRP-α D1 domain having the sequence of any one of SEQ ID NO:2. In some embodiments, the polypeptide of this aspect of the disclosureincludes no more than seven amino acid substitutions relative to thewild-type SIRP-α D1 domain having the sequence of any one of SEQ ID NO:2.

In some embodiments, the polypeptide binds CD47 with at least 10-foldgreater binding affinity than the wild-type SIRP-α D1 domain having thesequence of any one of SEQ ID NO: 2. In some embodiments, thepolypeptide binds CD47 with at least 100-fold greater binding affinitythan the wild-type SIRP-α D1 domain having the sequence of any one ofSEQ ID NO: 2. In some embodiments, the polypeptide binds CD47 with atleast 1000-fold greater binding affinity than the wild-type SIRP-α D1domain having the sequence of any one of SEQ ID NO: 2. In someembodiments, fragments include polypeptides of less than 10 amino acidsin length, about 10 amino acids in length, about 20 amino acids inlength, about 30 amino acids in length, about 40 amino acids in length,about 50 amino acids in length, about 60 amino acids in length, about 70amino acids in length, about 80 amino acids in length, about 90 aminoacids in length, about 100 amino acids in length, or more than about 100amino acids in length. Fragments retain the ability to bind to CD47.Preferably, SIRP-α D1 variant polypeptides and fragments thereof bind toCD47 with a higher affinity than a SIRP-α polypeptide binds to CD47. Forexample, in some embodiments, a SIRP-α D1 variant polypeptide orfragment thereof binds to CD47 with a K_(D) less than 1×10⁻⁸ M, lessthan 5×10⁻⁹ M, less than 1×10⁻⁹ M, less 5×10⁻¹⁰ M, less than 1×10⁻¹⁰ Mor less than 1×10⁻¹¹M. In some embodiments, a SIRP-α D1 variantpolypeptide or fragment thereof binds to CD47 with a K_(D) between about500 nM and 100 nM, between about 100 nM and 50 nM, between about 50 nMand 10 nM, between about 10 nM and 5 nM, between about 5 nM and 1 nM,between about 1 nM and 500 pM, between about 500 pM and 100 pM, betweenabout 100 pM and 50 pM, or between about 50 pM and 10 pM.

Described herein, in some embodiments, is a polypeptide comprising aSIRP-α D1 variant having a sequence according to:

EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅X₆GX₇FPRVTTVSDX₈TKRNNMDFSIRIGX₉X₁₀X₁₁X₁₂ADAGTYYCX₁₃KFRKGSPDDVEFKSGAGTELS VRAKPS(SEQ ID NO: 218), wherein X₁ is V, L, or I; X₂ is A, V, L, or I; X₃ isI, S, T, or F; X₄ is E, L, or V; X₅ is K or R; X₆ is E or Q; X₇ is H, R,or P; X₈ is S, G, L, or T; X₉ is any amino acid; X₁₀ is any amino acid;X₁₁ is any amino acid; X₁₂ is any amino acid; and X₁₃ is V or I; andwherein the SIRP-α D1 variant has at least two amino acid substitutionsrelative to a wild-type SIRP-α D1 domain having a sequence according toSEQ ID NO: 1.

In some embodiments, the polypeptide has the sequence of SEQ ID NO: 212,wherein X₁, wherein X₉ is A. In any of the aforementioned embodiments inthis aspect of the disclosure, X₉ is N. In any of the aforementionedembodiments in this aspect of the disclosure X₁₀ is I. In any of theaforementioned embodiments in this aspect of the disclosure X₉ is N andX10 is P. In any of the aforementioned embodiments in this aspect of thedisclosure X₉ is N and X11 is any amino acid other than S, T, or C. Inany of the aforementioned embodiments in this aspect of the disclosureX₁₁ is T. In any of the aforementioned embodiments in this aspect of thedisclosure X₁₁ is an amino acid other than T. In any of theaforementioned embodiments in this aspect of the disclosure X₁₂ is P. Inany of the aforementioned embodiments in this aspect of the disclosureX₉ is N and X₁₂ is any amino acid other than P.

Described herein, in some embodiments, is a polypeptide comprising aSIRP-α D1 variant having a sequence according to:

EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅X₆GX₇FPRVTTVSDX₈TKRNNMDFSIRIGX₉ITX₁₀ADAGTYYCX₁₁KFRKGSPDDVEFKSGAGTELSVRA KPS(SEQ ID NO: 219), wherein X₁ is V, L, or I; X₂ is A, V, L, or I; X₃ isI, S, T, or F; X₄ is E, L, or V; X₅ is K or R; X₆ is E or Q; X₇ is H, R,or P; X₈ is S, G, L, or T; X₉ is N; X₁₀ is any amino acid other than P;and X₁₁ is V or I; and wherein the SIRP-α D1 variant has at least twoamino acid substitutions relative to a wild-type SIRP-α D1 domain havinga sequence according to SEQ ID NO: 1.

In another aspect of the disclosure, compositions are disclosed hereinwhich include a SIRP-α D1 variant polypeptide having the amino acidsequence of SEQ ID NO: 48, or a fragment thereof. In some embodiments,the SIRP-α D1 variant polypeptide or fragment thereof binds to CD47 witha higher affinity compared to the affinity that a SIRP-α polypeptidebinds to the CD47. In some embodiments, the SIRP-α D1 variantpolypeptide binds to CD47 with a K_(D) less than 1×10⁻⁸M, or less than1×10⁻⁹M, less than 1×10⁻¹⁰M or less than 1×10⁻¹¹M. In some embodiments,the above-mentioned SIRP-α D1 variant polypeptides are attached or fusedto a second polypeptide. In some embodiments, the second polypeptideincludes, without limitation, an Fc polypeptide, an Fc variant, an HSApolypeptide, an albumin peptide, a PEG polymer or a fragment of theforegoing.

Without limiting the foregoing, in some embodiments, a SIRP-α D1 variantpolypeptide is selected from any one of SEQ ID NOs: 53-87 and 213 shownin Table 6.

TABLE 6 SIRP-α Variant Polypeptides SEQ ID NO: Amino Acid Sequence  53EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFK SGAGTELSVRAKPS  54EEELQVIQPDKSVSVAAGESAILHCTVTSLFPVGPIQWFRGAGPARELIYNQRQGPFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFK SGAGTELSVRAKPS  55EEELQVIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFK SGAGTELSVRAKPS  56EEELQIIQPDKSVSVAAGESAILHCTVTSLFPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFK SGAGTELSVRAKPS  57EEELQIIQPDKSVSVAAGESAILHCTITSLIPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFK SGAGTELSVRAKPS  58EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARELIYNQRQGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFK SGAGTELSVRAKPS  59EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQKQGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFK SGAGTELSVRAKPS  60EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQREGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFK SGAGTELSVRAKPS  61EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQRQGHFPRVTTVSETTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFK SGAGTELSVRAKPS  62EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTVSESTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFK SGAGTELSVRAKPS  63EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFK SGAGTELSVRAKPS  64EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQREGPFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFK SGAGTELSVRAKPS  65EEELQVIQPDKSVSVAAGESAILHCTVTSLFPVGPIQWFRGAGPARELIYNQREGPFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFK SGAGTELSVRAKPS  66EEELQVIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARELIYNQREGPFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFK SGAGTELSVRAKPS  67EEELQVIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARELIYNQREGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFK SGAGTELSVRAKPS  68EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARELIYNQREGPFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFK SGAGTELSVRAKPS  69EEELQVIQPDKSVSVAAGESAILHCTITSLIPVGPIQWFRGAGPARELIYNQREGPFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFK SGAGTELSVRAKPS  70EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARELIYNQREGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFK SGAGTELSVRAKPS  71EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEF KSGAGTELSVRAKPS  72EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEF KSGAGTELSVRAKPS  73EEELQVIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEF KSGAGTELSVRAKPS  74EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEF KSGAGTELSVRAKPS  75EEELQVIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEF KSGAGTELSVRAKPS  76EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEF KSGAGTELSVRAKPS  77EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEF KSGAGTELSVRAKPS  78EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCIKFRKGSPDDVEF KSGAGTELSVRAKPS  79EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEF KSGAGTELSVRAKPS  80EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCIKFRKGSPDDVEF KSGAGTELSVRAKPS  81EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEF KSGAGTELSVRAKPS  82EEELQVIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEF KSGAGTELSVRAKPS  83EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEF KSGAGTELSVRAKPS  84EEELQVIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEF KSGAGTELSVRAKPS  85EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEF KSGAGTELSVRAKPS  86EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEF KSGAGTELSVRAKPS  87EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEF KSGAGTELSVRAKPS 213EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGNITVADAGTYYCVKFRKGSPDDVEF KSGAGTELSVRAKPS

In some embodiments, the polypeptide includes a high affinity SIRP-α D1domain that has at least 85% sequence identity (e.g., at least 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity) to any variant provided in Table 6.

In some embodiments, the polypeptide includes a high affinity SIRP-α D1domain that has at least 85% sequence identity (e.g., at least 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity) to SEQ ID NOs: 80, 81, or 85 in Table 6.

III. Fc Domain Variants and Fusion Constructs

Disclosed herein, in some embodiments, are polypeptides comprising asignal-regulatory protein α (SIRP-α) D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92.

Also disclosed herein, in some embodiments, are polypeptides comprisingan Fc variant, wherein the Fc variant comprises an Fc domain dimerhaving two Fc domain monomers, wherein each Fc domain monomerindependently is selected from (i) a human IgG1 Fc region consisting ofmutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc regionconsisting of mutations A330S, P331S and N297A; or (iii) a human IgG4 Fcregion comprising mutations S228P, E233P, F234V, L235A, delG236, andN297A.

Antibodies that target cell surface antigens can triggerimmunostimulatory and effector functions that are associated with Fcreceptor (FcR) engagement on immune cells. There are a number of Fcreceptors that are specific for particular classes of antibodies,including IgG (gamma receptors), IgE (eta receptors), IgA (alphareceptors) and IgM (mu receptors). Binding of the Fc region to Fcreceptors on cell surfaces can trigger a number of biological responsesincluding phagocytosis of antibody-coated particles (antibody-dependentcell-mediated phagocytosis, or ADCP), clearance of immune complexes,lysis of antibody-coated cells by killer cells (antibody-dependentcell-mediated cytotoxicity, or ADCC) and, release of inflammatorymediators, placental transfer, and control of immunoglobulin production.Additionally, binding of the C1 component of complement to antibodiescan activate the complement system. Activation of complement can beimportant for the lysis of cellular pathogens. However, the activationof complement can also stimulate the inflammatory response and can alsobe involved in autoimmune hypersensitivity or other immunologicaldisorders. Variant Fc regions with reduced or ablated ability to bindcertain Fc receptors are useful for developing therapeutic antibodiesand Fc-fusion polypeptide constructs which act by targeting, activating,or neutralizing ligand functions while not damaging or destroying localcells or tissues.

In some embodiments, a SIRP-α D1 polypeptide construct comprises anon-naturally occurring high affinity SIRP-α D1 variant linked to an Fcdomain monomer which forms an Fc domain having ablated or reducedeffector function.

In some embodiments, a Fc domain monomer refers to a polypeptide chainthat includes second and third antibody constant domains (e.g., CH2 andCH3). In some embodiments, an Fc domain monomer also includes a hingedomain. In some embodiments, the Fc domain monomer is of anyimmunoglobulin antibody isotype, including IgG, IgE, IgM, IgA, and IgD.Additionally, in some embodiments, an Fc domain monomer is of any IgGsubtype (e.g., IgG1, IgG2, IgG2a, IgG2b, IgG2c, IgG3, and IgG4). In someembodiments, Fc domain monomers include as many as ten changes from awild-type Fc domain monomer sequence (e.g., 1-10, 1-8, 1-6, 1-4 aminoacid substitutions, additions or insertions, deletions, or combinationsthereof) that alter the interaction between an Fc domain and an Fcreceptor.

As used herein, the term “Fc domain” refers to a dimer of two Fc domainmonomers. In a wild-type Fc domain, two Fc domain monomers dimerize bythe interaction between the two CH3 antibody constant domains, as wellas one or more disulfide bonds that form between the hinge domains ofthe two dimerized Fc domain monomers. In some embodiments, an Fc domainis mutated to lack effector functions, for example a “dead Fc domain.”In some embodiments, each of the Fc domain monomers in an Fc domainincludes amino acid substitutions in the CH2 antibody constant domain toreduce the interaction or binding between the Fc domain and an Fcreceptor, such as an Fcγ receptor (FcγR), an Fcα receptor (FcαR), or anFcε (FcεR).

In some embodiments, a high affinity SIRP-α D1 variant (e.g., any of thevariants described in Tables 2, 5, and 6) is fused to an Fc domainmonomer of an immunoglobulin or a fragment of an Fc domain monomer. Insome embodiments, an Fc domain monomer of an immunoglobulin or afragment of an Fc domain monomer is capable of forming an Fc domain withanother Fc domain monomer. In some embodiments, an Fc domain monomer ofan immunoglobulin or a fragment of an Fc domain monomer is not capableof forming an Fc domain with another Fc domain monomer. In someembodiments, an Fc domain monomer or a fragment of an Fc domain is fusedto a polypeptide of the disclosure to increase serum half-life of thepolypeptide. In some embodiments, an Fc domain monomer or a fragment ofan Fc domain monomer fused to a polypeptide of the disclosure dimerizeswith a second Fc domain monomer to form an Fc domain which binds an Fcreceptor, or alternatively, an Fc domain monomer binds to an Fcreceptor. In some embodiments, an Fc domain or a fragment of the Fcdomain fused to a polypeptide to increase serum half-life of thepolypeptide does not induce any immune system-related response.

In some embodiments, a SIRP-α polypeptide or construct provided hereinincludes a SIRP-α D1 domain or variant thereof joined to a first Fcdomain monomer and an antibody variable domain joined to a second Fcdomain monomer, in which the first and second Fc domain monomers combineto form an Fc domain (e.g., a heterodimeric Fc domain). An Fc domain isthe protein structure that is found at the C-terminus of animmunoglobulin. An Fc domain includes two Fc domain monomers that aredimerized by the interaction between the CH3 antibody constant domains.A wild-type Fc domain forms the minimum structure that binds to an Fcreceptor, e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and FcγRIV.

The Fc domain is not involved directly in binding an antibody to itstarget, but can be involved in various effector functions, such asparticipation of the antibody in antibody-dependent cellular toxicity.In some embodiments, the Fc domain in a SIRP-α polypeptide or constructof the disclosure comprise amino acid substitutions, additions orinsertions, deletions, or any combinations thereof that lead todecreased effector function such as decreased antibody-dependentcell-mediated cytotoxicity (ADCC), decreased complement-dependentcytolysis (CDC), decreased antibody-dependent cell-mediated phagocytosis(ADCP), or any combinations thereof. In some embodiments, the SIRP-αpolypeptides or constructs of the disclosure are characterized bydecreased binding (e.g., minimal binding or absence of binding) to ahuman Fc receptor and decreased binding (e.g., minimal binding orabsence of binding) to complement protein C1q. In some embodiments, theSIRP-α constructs of the disclosure are characterized by decreasedbinding (e.g., minimal binding or absence of binding) to human FcγRI,FcγRIIA, FcγRIIB, FcγRIIIB, FcγRIIIB, or any combinations thereof, andC1q. To alter or reduce an antibody-dependent effector function, such asADCC, CDC, ADCP, or any combinations thereof, in some embodiments, theFc domains in SIRP-α constructs of the disclosure are of the IgG classand comprise one or more amino acid substitutions at E233, L234, L235,G236, G237, D265, D270, N297, E318, K320, K322, A327, A330, P331, orP329 (numbering according to the EU index of Kabat (Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991))).

In some embodiments, polypeptide constructs comprising a non-native Fcregion described herein exhibit reduced or ablated binding to at leastone of Fcγ receptors CD16a, CD32a, CD32b, CD32c, and CD64 as compared toa polypeptide construct comprising a native Fc region. In some cases,the polypeptide constructs described herein exhibit reduced or ablatedbinding to CD16a, CD32a, CD32b, CD32c, and CD64 Fcγ receptors.

CDC refers to a form of cytotoxicity in which the complement cascade isactivated by the complement component C1q binding to antibody Fc. Insome embodiments, polypeptide constructs comprising a non-native Fcregion described herein exhibit at least a 5%, 10%, 15%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% or greater reduction in C1q binding compared toa polypeptide construct comprising a wild-type Fc region. In some cases,polypeptide constructs comprising a non-native Fc region as describedherein exhibit reduced CDC as compared to a polypeptide constructcomprising a wild-type Fc region. In some embodiments, polypeptideconstructs comprising a non-native Fc region as described herein exhibitat least a 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% orgreater reduction in CDC compared to a polypeptide construct comprisinga wild-type Fc region. In some cases, polypeptide constructs comprisinga non-natural Fc variant as described herein exhibit negligible CDC ascompared to a polypeptide construct comprising a wild-type Fc region.

In some embodiments, the Fc variants herein are minimally glycosylatedor have reduced glycosylation relative to a wild-type sequence. In someembodiments, deglycosylation is accomplished with a mutation of N297A,or by mutating N297 to any amino acid which is not N. In someembodiments, deglycosylation is accomplished by disrupting the motifN-Xaa1-Xaa2-Xaa3, wherein N=asparagine; Xaa1=any amino acid except P(proline); Xaa2=T (threonine), S (serine) or C (cysteine); and Xaa3=anyamino acid except P (proline). In one embodiment, the N-Xaa1-Xaa2-Xaa3motif refers to residues 297-300 as designated according to Kabat etal., 1991. In some embodiments, a mutation to any one or more of N,Xaa1, Xaa2, or Xaa3 results in deglycosylation of the Fc variant.

In some embodiments, variants of antibody IgG constant regions (e.g., Fcvariants) possess a reduced capacity to specifically bind Fcγ receptorsor have a reduced capacity to induce phagocytosis. In some embodiments,variants of antibody IgG constant regions (e.g., Fc variants) possess areduced capacity to specifically bind Fcγ receptors and have a reducedcapacity to induce phagocytosis. For example, in some embodiments, an Fcdomain is mutated to lack effector functions, typical of a “dead” Fcdomain. For example, in some embodiments, an Fc domain includes specificamino acid substitutions that are known to minimize the interactionbetween the Fc domain and an Fcγ receptor. In some embodiments, an Fcdomain monomer is from an IgG1 antibody and includes one or more ofamino acid substitutions L234A, L235A, G237A, and N297A (as designatedaccording to the EU numbering system per Kabat et al., 1991). In someembodiments, one or more additional mutations are included in such IgG1Fc variant. Non-limiting examples of such additional mutations for humanIgG1 Fc variants include E318A and K322A. In some instances, a humanIgG1 Fc variant has up to 12, 11, 10, 9, 8, 7, 6, 5 or 4 or fewermutations in total as compared to wild-type human IgG1 sequence. In someembodiments, one or more additional deletions are included in such IgG1Fc variant. For example, in some embodiments, the C-terminal lysine ofthe Fc IgG1 heavy chain constant region provided in SEQ ID NO: 88 inTable 7 is deleted, for example to increase the homogeneity of thepolypeptide when the polypeptide is produced in bacterial or mammaliancells. In some instances, a human IgG1 Fc variant has up to 12, 11, 10,9, 8, 7, 6, 5 or 4 or fewer deletions in total as compared to wild-typehuman IgG1 sequence. In some embodiments, a IgG1 Fc variant has asequence according to any one of SEQ ID NO: 135, SEQ ID NO: 136, or SEQID NO: 137.

In some embodiments, an Fc domain monomer is from an IgG2 or IgG4antibody and includes amino acid substitutions A330S, P331S, or bothA330S and P331S. The aforementioned amino acid positions are definedaccording to Kabat, et al. (1991). The Kabat numbering of amino acidresidues can be determined for a given antibody by alignment at regionsof homology of the sequence of the antibody with a “standard” Kabatnumbered sequence. In some embodiments, the Fc variant comprises a humanIgG2 Fc sequence comprising one or more of A330S, P331S and N297A aminoacid substitutions (as designated according to the EU numbering systemper Kabat, et al. (1991). In some embodiments, one or more additionalmutations are included in such IgG2 Fc variants. Non-limiting examplesof such additional mutations for human IgG2 Fc variant include V234A,G237A, P238S, V309L and H268A (as designated according to the EUnumbering system per Kabat et al. (1991)). In some instances, a humanIgG2 Fc variant has up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or fewermutations in total as compared to wild-type human IgG2 sequence. In someembodiments, one or more additional deletions are included in such IgG2Fc variant. For example, in some embodiments, the C-terminal lysine ofthe Fc IgG2 heavy chain constant region provided in SEQ ID NO: 89 inTable 7 is deleted, for example to increase the homogeneity of thepolypeptide when the polypeptide is produced in bacterial or mammaliancells. In some instances, a human IgG2 Fc variant has up to 12, 11, 10,9, 8, 7, 6, 5 or 4 or fewer deletions in total as compared to wild-typehuman IgG2 sequence.

When the Fc variant is an IgG4 Fc variant, in some embodiments, such Fcvariant comprises a S228P mutation (as designated according to Kabat, etal. (1991)). In some instances, a human IgG4 Fc variant has up to 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutation(s) in total as compared towild-type human IgG4 sequence.

In some embodiments, the Fc variant includes at least one of themutations L234A, L235A, G237A or N297A of an IgG1 Fc region or at leastone of the mutations A330S, P331S or N297A of an IgG2 Fc region. In someembodiments, the Fc variant includes at least two of the mutationsL234A, L235A, G237A or N297A of an IgG1 Fc region or at least two of themutations A330S, P331S or N297A of an IgG2 Fc region. In someembodiments, the Fc variant includes at least three of the mutationsL234A, L235A, G237A or N297A of an IgG1 Fc region or consists of themutations A330S, P331S and N297A of an IgG2 Fc region. In someembodiments, the Fc variant consists of the mutations L234A, L235A,G237A and N297A.

In some embodiments, the Fc variant exhibits reduced binding to an Fcreceptor of the subject compared to the wild-type human IgG Fc region.In some embodiments, the Fc variant exhibits ablated binding to an Fcreceptor of the subject compared to the wild-type human IgG Fc region.In some embodiments, the Fc variant exhibits a reduction of phagocytosiscompared to the wild-type human IgG Fc region. In some embodiments, theFc variant exhibits ablated phagocytosis compared to the wild-type humanIgG Fc region.

SEQ ID NO: 88 and SEQ ID NO: 89 provide amino acid sequences of Fc IgG1and IgG2 heavy chain constant regions. In some embodiments, an Fcvariant is any variant of SEQ ID NOs: 90-95 as shown in Table 7.

TABLE 7 Amino Acid Sequences of Fc Variants SEQ ID NO:Amino Acid Sequence 88 EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 89STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 90DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 91DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 92VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK93 VECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGEYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG94 ERKSSVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGEYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 95ERKSSVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGEYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Antibody-dependent cell-mediated cytotoxicity, which is also referred toherein as ADCC, refers to a form of cytotoxicity in which secreted Igbound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g.,Natural Killer (NK) cells and neutrophils) enabling these cytotoxiceffector cells to bind specifically to an antigen-bearing target celland subsequently kill the target cell. Antibody-dependent cell-mediatedphagocytosis, which is also referred to herein as ADCP, refers to a formof cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain phagocytic cells (e.g., macrophages) enabling thesephagocytic effector cells to bind specifically to an antigen-bearingtarget cell and subsequently engulf and digest the target cell.Ligand-specific high-affinity IgG antibodies directed to the surface oftarget cells can stimulate the cytotoxic or phagocytic cells and can beused for such killing. In some embodiments, polypeptide constructscomprising an Fc variant as described herein exhibit reduced ADCC orADCP as compared to a polypeptide construct comprising a wild-type Fcregion. In some embodiments, polypeptide constructs comprising an Fcvariant as described herein exhibit at least a 5%, 10%, 15%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90% or greater reduction in ADCC or ADCPcompared to a polypeptide construct comprising a wild-type Fc region. Insome embodiments, polypeptide constructs comprising an Fc variant asdescribed herein exhibit ablated ADCC or ADCP as compared to apolypeptide construct comprising a wild-type Fc region.

Complement-directed cytotoxicity, which is also referred to herein asCDC, refers to a form of cytotoxicity in which the complement cascade isactivated by the complement component C1q binding to antibody Fc. Insome embodiments, polypeptide constructs comprising an Fc variant asdescribed herein exhibit at least a 5%, 10%, 15%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% or greater reduction in C1q binding compared to apolypeptide construct comprising a wild-type Fc region. In some cases,polypeptide constructs comprising an Fc variant as described hereinexhibit reduced CDC as compared to a polypeptide construct comprising awild-type Fc region. In some embodiments, polypeptide constructscomprising an Fc variant as described herein exhibit at least a 5%, 10%,15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater reduction in CDCcompared to a polypeptide construct comprising a wild-type Fc region. Insome cases, polypeptide constructs comprising an Fc variant as describedherein exhibit negligible CDC as compared to a polypeptide constructcomprising a wild-type Fc region.

Fc variants herein include those that exhibit reduced binding to an Fcγreceptor compared to the wild-type human IgG Fc region. For example, insome embodiments, an Fc variant exhibits binding to an Fcγ receptor thatis less than the binding exhibited by a wild-type human IgG Fc region toan Fcγ receptor, as described in the Examples. In some instances, an Fcvariant has reduced binding to an Fcγ receptor by a factor of 10%, 20%30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%(fully ablated effector function). In some embodiments, the reducedbinding is for any one or more Fcγ receptor, e.g., CD16a, CD32a, CD32b,CD32c, or CD64.

In some instances, the Fc variants disclosed herein exhibit a reductionof phagocytosis compared to its wild-type human IgG Fc region. Such Fcvariants exhibit a reduction in phagocytosis compared to its wild-typehuman IgG Fc region, wherein the reduction of phagocytosis activity ise.g., by a factor of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,96%, 97%, 98%, 99% or 100%. In some instances, an Fc variant exhibitsablated phagocytosis compared to its wild-type human IgG Fc region.

In some embodiments, the Fc variants disclosed herein are coupled to oneor more fusion partners. In some cases the fusion partner is atherapeutic moiety. In some cases, the fusion partner is selected toenable targeting of an expressed protein, purification, screening,display, and the like. In some embodiments, the fusion partner alsoaffects the degree of binding to Fc receptors or the degree ofphagocytosis reduction. As described herein, in some embodiments, whenan Fc variant is coupled to a fusion partner, it forms a polypeptideconstruct as described below.

In some embodiments, fusion partners are linked to the Fc variantsequence via a linker sequence. In some embodiments, the linker sequencegenerally comprises a small number of amino acids, such as less than tenamino acids, although longer linkers are also utilized. In some cases,the linker has a length less than 10, 9, 8, 7, 6, or 5 amino acids orshorter. In some cases, the linker has a length of at least 10, 11, 12,13, 14, 15, 20, 25, 30, or 35 amino acids or longer. Optionally, in someembodiments, a cleavable linker is employed.

In some embodiments, a fusion partner is a targeting or signal sequencethat directs an Fc variant protein and any associated fusion partners toa desired cellular location or to the extracellular media. In someembodiments, certain signaling sequences target a protein to be eithersecreted into the growth media, or into the periplasmic space, locatedbetween the inner and outer membrane of the cell. In some embodiments, afusion partner is a sequence that encodes a peptide or protein thatenables purification or screening. Such fusion partners include, but arenot limited to, polyhistidine tags (His-tags) (for example His6 andHis10) or other tags for use with Immobilized Metal AffinityChromatography (IMAC) systems (e.g., Ni+2 affinity columns), GSTfusions, MBP fusions, Strep-tag, the BSP biotinylation target sequenceof the bacterial enzyme BirA, and epitope tags which are targeted byantibodies (for example c-myc tags, flag-tags, and the like).

In some embodiments, such tags are useful for purification, forscreening, or both. For example, in some embodiments, an Fc variant ispurified using a His-tag by immobilizing it to a Ni+2 affinity column,and then after purification the same His-tag is used to immobilize theantibody to a Ni+2 coated plate to perform an ELISA or other bindingassay as described elsewhere herein. In some embodiments, a fusionpartner enables the use of a selection method to screen Fc variants asdescribed herein.

Various fusion partners that enable a variety of selection methods areavailable. For example, by fusing the members of an Fc variant libraryto the gene III protein, phage display can be employed. In someembodiments, fusion partners enable Fc variants to be labeled.Alternatively, in some embodiments, a fusion partner binds to a specificsequence on the expression vector, enabling the fusion partner andassociated Fc variant to be linked covalently or noncovalently with thenucleic acid that encodes them.

In some embodiments, when a fusion partner is a therapeutic moiety, thetherapeutic moiety is, e.g., a peptide, a protein, an antibody, a siRNA,or a small molecule. Non-limiting examples of therapeutic antibodiesthat are coupled to the Fc variants of the present disclosure include,but are not limited to antibodies that recognize CD47. Non-limitingexamples of therapeutic polypeptides that are coupled to the Fc variantsof the present disclosure include, but are not limited to, CD47 bindingpolypeptides, including SIRP-α polypeptides. In such instances, the CD47binding polypeptide is attached or fused to an Fc variant of thedisclosure. Examples of CD47 binding polypeptides include, but are notlimited to, anti-CD47 antibodies or fragments thereof, and ligands ofCD47 such as SIRP-α or a fragment thereof. Additional examples of CD47binding polypeptides include, but are not limited to naturally-occurringforms of SIRP-α as well as mutants thereof.

In some embodiments, disclosed herein is a polypeptide comprising an Fcvariant, wherein the Fc variant comprises an Fc domain dimer having twoFc domain monomers, wherein each Fc domain monomer independently isselected from (i) a human IgG1 Fc region consisting of mutations L234A,L235A, G237A, and N297A; (ii) a human IgG2 Fc region consisting ofmutations A330S, P331S and N297A; or (iii) a human IgG4 Fc regioncomprising mutations S228P, E233P, F234V, L235A, delG236, and N297A. Insome embodiments, the Fc domain monomers are identical (i.e.,homodimer). In some embodiments, the Fc domain monomers are different(i.e., heterodimer). In some embodiments, at least one of the Fc domainmonomers in an Fc domain dimer is a human IgG1 Fc region consisting ofmutations L234A, L235A, G237A, and N297A. In some embodiments, at leastone of the Fc domain monomers in an Fc domain dimer is a human IgG2 Fcregion consisting of mutations A330S, P331S and N297A. In someembodiments, the Fc variant exhibits ablated or reduced binding to anFcγ receptor compared to the wild-type version of the human IgG Fcregion. In some embodiments, the Fc variant exhibits ablated or reducedbinding to CD16a, CD32a, CD32b, CD32c, and CD64 Fcγ receptors comparedto the wild-type version of the human IgG Fc region. In someembodiments, the Fc variant exhibits ablated or reduced binding to C1qcompared to the wild-type version of the human IgG Fc fusion. In someembodiments, at least one of the Fc domain monomers in an Fc domaindimer is a human IgG4 Fc region comprising mutations S228P, E233P,F234V, L235A, delG236, and N297A. In some embodiments, the Fc variantexhibits ablated or reduced binding to a Fcγ receptor compared to thewild-type human IgG4 Fc region. In some embodiments, the Fc variantexhibits ablated or reduced binding to CD16a and CD32b Fcγ receptorscompared to the wild-type version of its human IgG4 Fc region. In someembodiments, the Fc variant binds to an Fcγ receptor with a K_(D)greater than about 5×10⁻⁶ M.

In some embodiments, the Fc variant further comprises a CD47 bindingpolypeptide. In some embodiments, the Fc variant exhibits ablated orreduced binding to an Fcγ receptor compared to a wild-type version of ahuman IgG Fc region. In some embodiments, the CD47 binding polypeptidedoes not cause acute anemia in rodents and non-human primates. In someembodiments, the CD47 binding polypeptide does not cause acute anemia inhumans.

In some embodiments, the CD47 binding polypeptide is a signal-regulatoryprotein α (SIRP-α) polypeptide or a fragment thereof. In someembodiments, the SIRP-α polypeptide comprises a SIRP-α D1 variantcomprising the amino acid sequence,EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQX₅EGX₆FPRVTTVSDX₇TKRNNMDFSIRIGX₈ITPADAGTYYCX₉KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ IDNO: 51), wherein X₁ is V or I; X₂ is A or I; X₃ is I or F; X₄ is E or V;X₅ is K or R; X₆ is H or P; X₇ is L or T; X₈ is any amino acid otherthan N; and X₉ is V or I. In some embodiments, the SIRP-α polypeptidecomprises a SIRP-α D1 variant wherein X₁ is V or I; X₂ is A or I; X₃ isI or F; X₄ is E; X₅ is K or R; X₆ is H or P; X₇ is L or T; X₈ is not N;and X₉ is V.

In some embodiments, disclosed herein, is a polypeptide comprising: aSIRP-α D1 variant, wherein the SIRP-α D1 variant is a non-naturallyoccurring high affinity SIRP-α D1 domain, wherein the SIRP-α D1 variantbinds to human CD47 with an affinity that is at least 10-fold greaterthan the affinity of a naturally occurring D1 domain; and an Fc domainmonomer, wherein the Fc domain monomer is linked to a second polypeptidecomprising a second Fc domain monomer to form an Fc domain, wherein theFc domain has ablated or reduced effector function. In some embodiments,the non-naturally occurring high affinity SIRP-α D1 domain comprises anamino acid mutation at residue 80.

In some embodiments, disclosed herein, is a SIRP-α D1 variant, whereinthe SIRP-α D1 variant binds CD47 from a first species with a K_(D) lessthan 250 nM; and wherein the SIRP-α D1 variant binds CD47 from a secondspecies with a K_(D) less than 250 nM; and the K_(D) for CD47 from thefirst species and the K_(D) for CD47 from the second species are within100 fold of each other; wherein the first species and the second speciesare selected from the group consisting of: human, rodent, and non-humanprimate. In some embodiments, the SIRP-α D1 variant binds CD47 from atleast 3 different species. In some embodiments, the non-human primate iscynomolgus monkey.

In some embodiments, disclosed herein, is a polypeptide comprising (a) aSIRP-α D1 domain that binds human CD47 with a K_(D) less than 250 nM;and (b) an Fc domain monomer linked to the N-terminus or the C-terminusof the SIRP-α D1 domain, wherein the polypeptide does not cause acuteanemia in rodents and non-human primates. In some embodiments, thepolypeptide is a non-naturally occurring variant of a human SIRP-α. Insome embodiments, administration of the polypeptide in vivo results inhemoglobin reduction by less than 50% during the first week afteradministration. In some embodiments, administration of the polypeptidein humans results in hemoglobin reduction by less than 50% during thefirst week after administration. In some embodiments, the polypeptidefurther comprises at least one Fc variant, wherein the Fc variantcomprises an Fc domain monomer selected from (i) a human IgG1 Fc regionconsisting of mutations L234A, L235A, G237A, and N297A; (ii) a humanIgG2 Fc region consisting of mutations A330S, P331S and N297A; or (iii)a human IgG4 Fc region comprising mutations S228P, E233P, F234V, L235A,delG236, and N297A. In some embodiments, the Fc domain monomer is ahuman IgG1 Fc region consisting of mutations L234A, L235A, G237A, andN297A. In some embodiments, the Fc domain monomer is a human IgG2 Fcregion consisting of mutations A330S, P331S and N297A.

The SIRP-α constructs of the disclosure include a SIRP-α domain orvariant thereof that has its C-terminus joined to the N-terminus of anFc domain monomer by way of a linker using conventional genetic orchemical means, e.g., chemical conjugation. In some embodiments, alinker (e.g., a spacer) is inserted between the polypeptide and the Fcdomain monomer. In some embodiments, a polypeptide of the disclosureincluding a high affinity SIRP-α D1 variant is fused to an Fc domainmonomer that is incapable of forming a dimer. In some embodiments, apolypeptide of the disclosure is fused to an Fc domain monomer that iscapable of forming a dimer, e.g., a heterodimer, with another Fc domainmonomer. In some embodiments, a polypeptide of the invention is fused toan Fc domain monomer and this fusion protein forms a homodimer. In someembodiments, a polypeptide of the disclosure is fused to a first Fcdomain monomer and a different protein or peptide (e.g., an antibodyvariable region) is fused to a second Fc domain monomer. In someembodiments, a SIRP-α D1 domain or variant thereof is joined to a firstFc domain monomer and a therapeutic protein (e.g., a cytokine, aninterleukin, an antigen, a steroid, an anti-inflammatory agent, or animmunomodulatory agent) is joined to a second Fc domain monomer. In someembodiments, the first and second Fc domain monomers form a heterodimer.

Without the limiting the foregoing, in some embodiments, a SIRP-α D1variant polypeptide (e.g., any of the variants described in Tables 2, 5,and 6) is fused to an Fc polypeptide or Fc variant polypeptide, such asan Fc domain monomer. Examples of polypeptides comprising a SIRP-α D1variant polypeptide and a fused Fc variant polypeptide include, but arenot limited to, SEQ ID NOS: 96-137, 214, and 216 shown in Table 8.

TABLE 8 Polypeptides Comprising SIRP-α D1 Variants Fused to Fc VariantsSEQ ID NO: Amino Acid Sequence  96EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK  97EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK  98EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK  99EEELQVIQPDKSVLVAAGETATLRCTATSLEPVGPIQWERGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 100EEELQVIQPDKSVLVAAGETATLRCTATSLEPVGPIQWERGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 101EEELQVIQPDKSVLVAAGETATLRCTITSLEPVGPIQWERGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 102EEELQIIQPDKSVLVAAGETATLRCTITSLEPVGPIQWERGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 103EEELQVIQPDKSVLVAAGETATLRCTITSLEPVGPIQWERGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 104EEELQIIQPDKSVLVAAGETATLRCTITSLEPVGPIQWERGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 105EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 106EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 107EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 108EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 109EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 110EEELQVIQPDKSVLVAAGETATLRCTITSLEPVGPIQWERGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 111EEELQIIQPDKSVLVAAGETATLRCTITSLEPVGPIQWERGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 112EEELQVIQPDKSVLVAAGETATLRCTITSLEPVGPIQWERGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 113EEELQIIQPDKSVLVAAGETATLRCTITSLEPVGPIQWERGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 114EEELQIIQPDKSVLVAAGETATLRCTITSLEPVGPIQWERGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 115EEELQVIQPDKSVLVAAGETATLRCTATSLEPVGPIQWERGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 116EEELQIIQPDKSVLVAAGETATLRCTITSLEPVGPIQWERGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 117EEELQVIQPDKSVLVAAGETATLRCTATSLEPVGPIQWERGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 118EEELQVIQPDKSVLVAAGETATLRCTATSLEPVGPIQWERGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 119EEELQVIQPDKSVLVAAGETATLRCTITSLEPVGPIQWERGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 120EEELQIIQPDKSVLVAAGETATLRCTITSLEPVGPIQWERGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 121EEELQVIQPDKSVLVAAGETATLRCTITSLEPVGPIQWERGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 122EEELQIIQPDKSVLVAAGETATLRCTITSLEPVGPIQWERGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 123EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 124EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 125EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 126EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSERKCCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 127EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSERKCCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 128EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSERKCCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 129EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSERKCCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 130EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPSESKYGPPCPSCPAPEFLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS LSLGK 131EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSESKYGPPCPPCPAPEFLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLGK 132EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSESKYGPPCPPCPAPEFEGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLGK 133EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSESKYGPPCPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS LSLGK 134EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSAAAPPCPPCPAPEFLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL SPGK 135EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG 136EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG 137EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG 214EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSERKSSVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 216EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK

In some embodiments, the polypeptide comprises a high affinity SIRP-α D1domain that has at least 85% sequence identity (e.g., at least 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity) to any variant provided in Table 8.

In some embodiments, the polypeptide comprises a high affinity SIRP-α D1domain that has at least 85% sequence identity (e.g., at least 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity) to SEQ ID NOs: 98-104, 107-113, 116-122, or 135-137in Table 8.

In some embodiments, the polypeptide comprises (a) a signal-regulatoryprotein α (SIRP-α) D1 variant, wherein the SIRP-α D1 variant comprisesthe amino acid sequence,EEX₁X₂QX₃IQPDKX₄VX₅VAAGEX₆X₇X₈LX₉CTX₁₀TSLX₁₁PVGPIQWFRGAGPX₁₂RX₁₃LIYNQX₁₄X₁₅GX₁₆FPRVTTVSX₁₇X₁₈TX₁₉RX₂₀NMDFX₂₁IX₂₂IX₂₃X₂₄ITX₂₅ADAGTYYCX₂₆KX₂₇RKGSPDX₂₈X₂₉EX₃₀KSGAGTELSVRX₃₁KPS (SEQ ID NO: 47), wherein X₁ is E, or G; X₂is L, I, or V; X₃ is V, L, or I; X₄ is S, or F; X₅ is L, or S; X₆ is S,or T; X₇ is A, or V; X₈ is I, or T; X₉ is H, R, or L; X₁₀ is A, V, I, orL; X₁₁ is I, T, S, or F; X₁₂ is A, or G; X₁₃ is E, V, or L; X₁₄ is K, orR; X₁₅ is E, or Q; X₁₆ is H, P, or R; X₁₇ is D, or E; X₁₈ is S, L, T, orG; X₁₉ is K, or R; X₂₀ is E, or N; X₂₁ is S, or P; X₂₂ is S, or R; X₂₃is S, or G; X₂₄ is any amino acid; X₂₅ is any amino acid; X₂₆ is V, orI; X₂₇ is F, L, or V; X₂₈ is D or absent; X₂₉ is T, or V; X₃₀ is F, orV; and X₃₁ is A, or G; and wherein the SIRP-α D1 variant has at leasttwo amino acid substitutions relative to a wild-type SIRP-α D1 domainhaving a sequence according to any one of SEQ ID NOs: 1 to 10; and (b)an Fc variant comprising an Fc domain dimer having two Fc domainmonomers, wherein each Fc domain monomer independently is (i) a humanIgG1 Fc region comprising a N297A mutation; (ii) a human IgG1 Fc regioncomprising L234A, L235A, and G237A mutations; (iii) a human IgG1 Fcregion comprising L234A, L235A, G237A, and N297A mutations; (iv) a humanIgG2 Fc region comprising a N297A mutation; (v) a human IgG2 Fc regioncomprising A330S and P331S mutations; (vi) a human IgG2 Fc regioncomprising A330S, P331S, and N297A mutations; (vii) a human IgG4 Fcregion comprising S228P, E233P, F234V, L235A, and delG236 mutations; or(viii) a human IgG4 Fc region comprising S228P, E233P, F234V, L235A,delG236, and N297A mutations.

In some embodiments, the polypeptide comprises a SIRP-α D1 variantwherein the SIRP-α D1 variant comprises an amino acid sequence accordingto SEQ ID NO: 47; an Fc variant comprising an Fc domain dimer having twoFc domain monomers, wherein one of the Fc domain monomers in the Fcdomain dimer comprises a human IgG1 Fc region comprising L234A, L235A,G237A, and N297A mutations.

Dimerization of Fc Domain Monomers

In some embodiments, a SIRP-α D1 variant polypeptide (e.g., any of thevariants described in Tables 2, 5, and 6) is fused to a first Fc domainmonomer either at the N-terminus or at the C-terminus. In someembodiments, the first Fc domain monomer is incapable of forming an Fcdomain or a dimer. In some embodiments, the first Fc domain monomercombines with a second Fc domain monomer to form an Fc domain or adimer. In some embodiments, the first and second Fc domain monomersinclude amino acid substitutions that promote heterodimerization betweenthe first and second domain monomers.

In some embodiments, each of the two Fc domain monomers in an Fc domainincludes amino acid substitutions that promote the heterodimerization ofthe two monomers. In some embodiments, a SIRP-α construct is formed, forexample, from a first subunit including a SIRP-α D1 variant polypeptidefused to a first Fc domain monomer and a second subunit including asecond Fc domain monomer (e.g., without a SIRP-α D1 variant polypeptideor any other polypeptide). In some embodiments, a construct has a singleSIRP-α D1 variant polypeptide linked to an Fc domain (e.g., single arm).In some embodiments, a construct has two SIRP-α D1 variant polypeptideslinked to an Fc domain (e.g., double arm). In some embodiments, a SIRP-αD1 variant having a K_(D) of about 500 nM is particularly useful in adouble arm construct. In some embodiments, a SIRP-α D1 variant having aK_(D) of about 50 nM is particularly useful in a double arm construct.In some embodiments, a SIRP-α D1 variant having a K_(D) of about 5 nM isuseful in a double arm construct and a single arm construct. In someembodiments, a SIRP-α D1 variant having a K_(D) of about 500 pM isuseful in a double arm construct and a single arm construct. In someembodiments, a SIRP-α D1 variant having a K_(D) of about 100 pM isuseful in a double arm construct and a single arm construct. In someembodiments, a SIRP-α D1 variant having a K_(D) of about 50 pM is usefulin a double arm construct and a single arm construct. In someembodiments, a SIRP-α D1 variant having a K_(D) of about 10 pM is usefulin a double arm construct and a single arm construct.

In some embodiments, heterodimerization of Fc domain monomers ispromoted by introducing different, but compatible, substitutions in thetwo Fc domain monomers, such as “knob-into-hole” residue pairs andcharge residue pairs. The knob and hole interaction favors heterodimerformation, whereas the knob-knob and the hole-hole interaction hinderhomodimer formation due to steric clash and deletion of favorableinteractions. A hole refers to a void that is created when an originalamino acid in a protein is replaced with a different amino acid having asmaller side-chain volume. A knob refers to a bump that is created whenan original amino acid in a protein is replaced with a different aminoacid having a larger side-chain volume. For example, in someembodiments, an amino acid being replaced is in the CH3 antibodyconstant domain of an Fc domain monomer and involved in the dimerizationof two Fc domain monomers. In some embodiments, a hole in one CH3antibody constant domain is created to accommodate a knob in another CH3antibody constant domain, such that the knob and hole amino acids act topromote or favor the heterodimerization of the two Fc domain monomers.In some embodiments, a hole in one CH3 antibody constant domain iscreated to better accommodate an original amino acid in another CH3antibody constant domain. In some embodiments, a knob in one CH3antibody constant domain is created to form additional interactions withoriginal amino acids in another CH3 antibody constant domain.

In some embodiments, a hole is constructed by replacing amino acidshaving larger side chains such as tyrosine or tryptophan with aminoacids having smaller side chains such as alanine, valine, or threonine,for example a Y407V mutation in the CH3 antibody constant domain.Similarly, in some embodiments, a knob is constructed by replacing aminoacids having smaller side chains with amino acids having larger sidechains, for example a T366W mutation in the CH3 antibody constantdomain. In some embodiments, one Fc domain monomer includes the knobmutation T366W and the other Fc domain monomer includes hole mutationsT366S, L358A, and Y407V. In some embodiments, a polypeptide of thedisclosure including a high affinity SIRP-α D1 variant is fused to an Fcdomain monomer including the knob mutation T366W to limit unwantedknob-knob homodimer formation. Examples of knob-into-hole amino acidpairs are included, without limitation, in Table 9 and examples ofknob-into-hole Fc variants and SIRP-α-Fc fusions are provided in Table10.

TABLE 9 Knob-into-Hole Amino Acid Pairs Fc domain Y407T Y407A F405AT394S T366S T394W T394S T366W monomer 1 L358A Y407T Y407A T394S Y407V Fcdomain T366Y T366W T394W F405W T366W T366Y T366W F405W monomer 2 F405AF405W Y407A

TABLE 10 Examples of Fc Variants and SIRP-α - Fc Fusions SEQ ID NO:Amino Acid Sequence 138 EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 139DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK 140EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 141DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK 142EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 143EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 145EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSEKTHTCPECPAPEAAGAPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 146EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 147DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK 148EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 149DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK

In addition to the knob-into-hole strategy, in some embodiments,electrostatic steering is also used to control the dimerization of Fcdomain monomers. Electrostatic steering refers to the utilization offavorable electrostatic interactions between oppositely charged aminoacids in peptides, protein domains, and proteins to control theformation of higher ordered protein molecules. In particular, to controlthe dimerization of Fc domain monomers using electrostatic steering, oneor more amino acid residues that make up the CH3-CH3 interface arereplaced with positively- or negatively-charged amino acid residues suchthat the interaction becomes electrostatically favorable or unfavorabledepending on the specific charged amino acids introduced. In someembodiments, a positively-charged amino acid in the interface, such aslysine, arginine, or histidine, is replaced with a negatively-chargedamino acid such as aspartic acid or glutamic acid. In some embodiments,a negatively-charged amino acid in the interface is replaced with apositively-charged amino acid. In some embodiments, the charged aminoacids are introduced to one of the interacting CH3 antibody constantdomains, or both. In some embodiments, introducing charged amino acidsto the interacting CH3 antibody constant domains of the two Fc domainmonomers promotes the selective formation of heterodimers of Fc domainmonomers as controlled by the electrostatic steering effects resultingfrom the interaction between charged amino acids. Examples ofelectrostatic steering amino acid pairs are included, withoutlimitation, in Table 11.

TABLE 11 Electrostatic Steering Amino Acid Pairs Fc domain K409D K409DK409E K409E K392D K392D K392E K392E K409D K370E monomer 1 K392D K409DK439E Fc domain D399K D399R D399K D399R D399K D399R D399K D399R D399KD356K monomer 2 D356K E357K D399K

Other methods used to control the heterodimerization of Fc domainmonomers, especially in the context of constructing a bispecificantibody, are available.

In some embodiments, a first Fc domain monomer and a second Fc domainmonomer each includes one or more of the following amino acidsubstitutions: T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T,Y349E, Y349V, L351T, L351H, L351N, L351K, P353S, S354D, D356K, D356R,D356S, E357K, E357R, E357Q, S364A, T366E, L368T, L368Y, L368E, K370E,K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T,D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E,K409D, K409T, and K409I, relative to the sequence of human IgG1.

In some embodiments an Fc domain monomer comprises: (a) one of thefollowing amino acid substitutions relative to wild type human IgG1:T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V,L351T, L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K,E357R, E357Q, S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q,K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R,D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T, orK409I; or (b) (i) a N297A mutation relative to a human IgG1 Fc region;(ii) a L234A, L235A, and G237A mutation relative to a human IgG1 Fcregion; (iii) a L234A, L235A, G237A, and N297A mutation relative to ahuman IgG1 Fc region; (iv) a N297A mutation relative to a human IgG2 Fcregion; (v) a A330S and P331S mutation relative to a human IgG2 Fcregion; (vi) a A330S, P331S, and N297A mutation relative to a human IgG2Fc region; (vii) a S228P, E233P, F234V, L235A, and delG236 mutationrelative to a human IgG4 Fc region; or (viii) a S228P, E233P, F234V,L235A, delG236, and N297A mutation relative to a human IgG4 Fc region.In some embodiments an Fc domain monomer comprises: (a) one of thefollowing amino acid substitutions relative to wild type human IgG1:T366W, T366S, L368A, Y407V, T366Y, T394W, F405W, Y349T, Y349E, Y349V,L351T, L351H, L351N, L351K, P353S, S354D, D356K, D356R, D356S, E357K,E357R, E357Q, S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q,K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R,D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T, orK409I; and (b) further comprises (i) a N297A mutation relative to ahuman IgG1 Fc region; (ii) a L234A, L235A, and G237A mutation relativeto a human IgG1 Fc region; (iii) a L234A, L235A, G237A, and N297Amutation relative to a human IgG1 Fc region; (iv) a N297A mutationrelative to a human IgG2 Fc region; (v) a A330S and P331S mutationrelative to a human IgG2 Fc region; (vi) a A330S, P331 S, and N297Amutation relative to a human IgG2 Fc region; (vii) a S228P, E233P,F234V, L235A, and delG236 mutation relative to a human IgG4 Fc region;or (viii) a S228P, E233P, F234V, L235A, delG236, and N297A mutationrelative to a human IgG4 Fc region.

In some embodiments, the first and second Fc domain monomers includedifferent amino acid substitutions. In some embodiments, the first Fcdomain monomer includes T366W. In some embodiments, the second Fc domainmonomer includes T366S, L368A, and Y407V. In some embodiments, the firstFc domain monomer includes D399K. In some embodiments, the second Fcdomain monomer includes K409D.

IV. Serum Albumin

Disclosed herein, in some embodiments, are polypeptides comprising asignal-regulatory protein α (SIRP-α) D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92.

Also disclosed herein, in some embodiments, are polypeptides comprisingan Fc variant, wherein the Fc variant comprises an Fc domain dimerhaving two Fc domain monomers, wherein each Fc domain monomerindependently is selected from (i) a human IgG1 Fc region consisting ofmutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc regionconsisting of mutations A330S, P331S and N297A; or (iii) a human IgG4 Fcregion comprising mutations S228P, E233P, F234V, L235A, delG236, andN297A.

Fusion to serum albumins can improve the pharmacokinetics of proteinpharmaceuticals, and in some embodiments, polypeptides of thedisclosure, including a high affinity SIRP-α D1 variant describedherein, is joined with a serum albumin.

Serum albumin is a globular protein that is abundant in blood inmammals. Serum albumin is produced in the liver and can constitute abouthalf of the blood serum proteins. It is monomeric and soluble in theblood. Some of the most crucial functions of serum albumin includetransporting hormones, fatty acids, and other proteins in the body,buffering pH, and maintaining osmotic pressure needed for properdistribution of bodily fluids between blood vessels and body tissues. Inpreferred embodiments, serum albumin is human serum albumin (HSA). Insome embodiments, an HSA is joined to the C-terminus of the polypeptideof the disclosure to increase the serum half-life of the polypeptide. Insome embodiments, the N-terminus of an HSA is joined to the C-terminusof the polypeptide of the disclosure. In some embodiments, a HSA isjoined, either directly or through a linker, to the C-terminus of thepolypeptide. In some embodiments, an HSA is joined, either directly orthrough a linker, to the N-terminus of the polypeptide.

In some embodiments, a human serum albumin comprises the sequence ofamino acids (aa) 25-609 of UniProt ID NO: P02768 (SEQ ID NO: 12) asshown in Table 12. In some embodiments, the HSA joined to a highaffinity SIRP-α D1 variant (e.g., any SIRP-α D1 variant described inTables 2, 5, and 6) includes amino acids 25-609 (SEQ ID NO: 12) of thesequence of UniProt ID NO: P02768. In some embodiments, the HSA includesC34S or K573P substitutions, relative to SEQ ID NO: 12. In someembodiments, the HSA includes C34S and K573P substitutions, relative toSEQ ID NO: 12.

TABLE 12 Sequence of HSA SEQ ID Descrip- NO: tion Amino Acid Sequence 12UniProt DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCP ID NO:FEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLF P02768,GDKLCTVATLRETYGEMADCCAKQEPERNECFLQ AA 25-HKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKY 609LYEIARRHPYFYAPELLFFAKRYKAAFTECCQAAD KAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVH TECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESK DVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVE EPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAED YLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQI KKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

In some embodiments, a serum albumin is fused genetically to apolypeptide of the disclosure or joined to the polypeptide throughchemical means, e.g., chemical conjugation. In some embodiments, aspacer is inserted between the polypeptide and the HSA. Some examples ofspacers are described in detail elsewhere herein. In some embodiments, aspacer is A or AAAL. In some embodiments, the fusion of an HSA in apolypeptide of the disclosure leads to prolonged retention of thepolypeptide as well as increases in half-life.

Polypeptides comprising a SIRP-α D1 variant polypeptide and a fused HSAinclude, but are not limited to, SEQ ID NOS: 150-159 provided in Table13.

TABLE 13 Polypeptides Comprising SIRP-α Variants Fused to HSA SEQ ID NO:Amino Acid Sequence 150 EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL 151EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL 152EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL 153EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL 154EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL 155EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL 156EEELQVIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL 157EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDLTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL 158EEELQVIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL 159EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

In some embodiments, the polypeptide includes a high affinity SIRP-α D1domain that has at least 85% sequence identity (e.g., at least 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity) to any variant provided in Table 13.

In some embodiments, the polypeptide includes a high affinity SIRP-α D1domain that has at least 85% sequence identity (e.g., at least 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity) to SEQ ID NO: 154, 155, and 159 in Table 13.

V. Albumin-Binding Peptide

Disclosed herein, in some embodiments, are polypeptides comprising asignal-regulatory protein α (SIRP-α) D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92.

Also disclosed herein, in some embodiments, are polypeptides comprisingan Fc variant, wherein the Fc variant comprises an Fc domain dimerhaving two Fc domain monomers, wherein each Fc domain monomerindependently is selected from (i) a human IgG1 Fc region consisting ofmutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc regionconsisting of mutations A330S, P331S and N297A; or (iii) a human IgG4 Fcregion comprising mutations S228P, E233P, F234V, L235A, delG236, andN297A.

Binding to serum proteins can improve the pharmacokinetics of proteinpharmaceuticals, and in particular, in some embodiments, thepolypeptides described herein are fused with serum protein-bindingpeptides or proteins.

As used herein, the term “albumin-binding peptide” refers to an aminoacid sequence of about 12 to 16 amino acids that has affinity for andfunctions to bind a serum albumin protein. In some embodiments, analbumin-binding peptide originates from human, mouse, or rat.

In some embodiments, a polypeptide of the disclosure including a highaffinity SIRP-α D1 variant (e.g., any variant provided in Tables 2, 5,and 6) is fused to an albumin-binding peptide that displays bindingactivity to serum albumin to increase the half-life of the polypeptide.Various albumin-binding peptides that can be used in the methods andcompositions described here are available. In some embodiments, thealbumin binding peptide includes the sequence DICLPRWGCLW (SEQ ID NO:160). In some embodiments, an albumin-binding peptide is fusedgenetically to a polypeptide of the disclosure or attached to thepolypeptide through chemical means, e.g., chemical conjugation.

In some embodiments, a linker (e.g., a spacer) is inserted between thepolypeptide and the albumin-binding peptide to allow for additionalstructural and spatial flexibility of the fusion protein. Specificlinkers (e.g., a spacer) and their amino acid sequences are described indetail further herein. In some embodiments, an albumin-binding peptideis fused to the N- or C-terminus of a polypeptide of the disclosure. Inone example, the N-terminus of the albumin-binding peptide is directlyfused to the C-terminus of a polypeptide of the disclosure through apeptide bond. In another example, the C-terminus of the albumin-bindingpeptide is directly fused to the N-terminus of a polypeptide of thedisclosure through a peptide bond. In some embodiments, the fusion of analbumin-binding peptide to a polypeptide of the disclosure leads toprolonged retention of the polypeptide through its binding to serumalbumin.

VI. Polyethylene Glycol (PEG) Polymer

Disclosed herein, in some embodiments, are polypeptides comprising asignal-regulatory protein α (SIRP-α) D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92.

Also disclosed herein, in some embodiments, are polypeptides comprisingan Fc variant, wherein the Fc variant comprises an Fc domain dimerhaving two Fc domain monomers, wherein each Fc domain monomerindependently is selected from (i) a human IgG1 Fc region consisting ofmutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc regionconsisting of mutations A330S, P331S and N297A; or (iii) a human IgG4 Fcregion comprising mutations S228P, E233P, F234V, L235A, delG236, andN297A.

In some embodiments, a polypeptide including a high affinity SIRP-α D1domain (e.g., any variant provided in Tables 2, 5, and 6) is fused to apolymer (e.g., polyethylene glycol, PEG). In some embodiments, theattachment of a polymer to a protein pharmaceutical “masks” the proteinpharmaceutical from the host's immune system. In addition, in someembodiments, certain polymers, such as hydrophilic polymers, providewater solubility to hydrophobic proteins and drugs. For example, in someembodiments, such polymers include PEG, polysialic acid chain, and PASchain molecules. In some embodiments, a polymer such as PEG, iscovalently attached to a cysteine substitution or addition in thepolypeptide. In some embodiments, the cysteine substitution in thepolypeptide is I7C, A16C, S20C, T20C, A45C, G45C, G79C, S79C, or A84C,relative to the sequence of any one of the sequences provided in Tables2, 5, and 6. In some embodiments, the addition of a cysteine residue inthe polypeptide is introduced using peptide synthesis, geneticmodification, molecular cloning, or any combinations thereof. In someembodiments, the polymer, for example PEG, is attached to the cysteineresidue using cysteine-maleimide conjugation. In some embodiments, apolymer such as PEG, is covalently attached to the polypeptide includinga high affinity SIRP-α D1 variant either at the N- or C-terminus or atan internal location, using conventional chemical methods such aschemical conjugation.

VII. Bispecific Construct

Disclosed herein, in some embodiments, are polypeptides comprising asignal-regulatory protein α (SIRP-α) D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92.

Also disclosed herein, in some embodiments, are polypeptides comprisingan Fc variant, wherein the Fc variant comprises an Fc domain dimerhaving two Fc domain monomers, wherein each Fc domain monomerindependently is selected from (i) a human IgG1 Fc region consisting ofmutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc regionconsisting of mutations A330S, P331S and N297A; or (iii) a human IgG4 Fcregion comprising mutations S228P, E233P, F234V, L235A, delG236, andN297A.

In some embodiments, a polypeptide having a high affinity SIRP-α D1variant (e.g., any of the variants provided in Tables 2, 5, and 6)comprises a bispecific construct. A bispecific construct refers to aconstruct that has two target-interacting domains. In some embodiments,a bispecific construct includes an Fc domain and two target-interactingdomains: (1) a SIRP-α D1 domain or variant thereof (e.g., any of thevariants provided in Tables 2, 5, and 6) and (2) an antibody variabledomain. In some embodiments, a bispecific construct includes a firstpolypeptide and a second polypeptide. In some embodiments, the firstpolypeptide has the formula A-L-B, wherein A includes a SIRP-α D1 domainor variant thereof, L is a linker, and B includes a first Fc domainmonomer. In some embodiments, the second polypeptide has the formulaA′-L′-B′, wherein A′ includes an antibody variable domain, L′ is alinker; and B′ includes a second Fc domain monomer. In some embodiments,the orientation of the first and second polypeptides is B-L-A andB′-L′-A′, respectively. In some embodiments, the first and second Fcdomain monomers combine to form the Fc domain in the bispecificconstruct. In some embodiments, a bispecific construct is of anyimmunoglobulin antibody isotypes (e.g., IgG, IgE, IgM, IgA, and IgD). Avariant of a SIRP-α D1 domain includes the D domain of a wild-type humanSIRP-α and one or more amino acid substitutions relative to thewild-type D1 domains (e.g., any SIRP-α D1 variant as described in Tables2, 5, and 6). In some embodiments, a SIRP-α D1 variant binds with higherbinding affinity to CD47 than does a wild-type human SIRP-α D1 domain.In some embodiments, the antibody variable domain in a bispecificconstruct targets a cell antigen (e.g., a cell antigen on a cancercell).

An antibody variable domain refers to the portions of the light andheavy chains of an antibody that include amino acid sequences ofcomplementary determining regions (CDRs, e.g., CDR L1, CDR L2, CDR L3,CDR H1, CDR H2, and CDR H3) and framework regions (FRs). The variabledomain of the antibody can confer on the antibody the ability to bind tospecific antigens. Many different antibody variable domain molecules canbe constructed. In some embodiments, the antibody variable domainmolecules used includes, but is not limited to, single-chain Fv.

In some embodiments, the antibody variable domain in a bispecificconstruct targets a cell antigen (e.g., a cell antigen on a cancer cellor on an immune cell). Some proteins are expressed at higher levels incancer cells than in non-cancer cells. For example, a cancer antigen isa protein that is expressed preferentially by cancer cells (e.g., it isexpressed at higher levels on cancer cells than on non-cancer cells) andin some instances it is expressed solely by cancer cells. In someembodiments, proteins, e.g., proteins expressed by cancer cells, thatare targeted by an antibody variable domain forming an Fc domain with ahigh affinity SIRP-α domain or variant thereof include, but are notlimited to: 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6,CD123, CD19, CD20, CD22, EpCAM, CD30, CD32b, CD33, CD37, CD38, CD40,CD52, CD70, CD74, CD79b, CD98, CEA, CEACAM5, CLDN18.2, CLDN6, CS1,CXCR4, DLL-4, EGFR, EGP-1, ENPP3, EphA3, ETBR, FGFR2, fibronectin,FR-alpha, GCC, GD2, glypican-3, GPNMB, HER-2, HER3, HLA-DR, ICAM-1,IGF-1R, IL-3R, LIV-1, mesothelin, MUC16, MUC1, NaPi2b, Nectin-4, Notch2, Notch 1, PD-L1, PD-L2, PDGFR-a, PS, PSMA, SLTRK6, STEAP1, TEM1,VEGFR, CD25, CD27L, DKK-1, or CSF-1R. In some embodiments, the antibodyvariable domain in the bispecific construct is not engineered to bind ahuman protein.

In some embodiments, each of the first and second Fc domain monomers inthe Fc domain of the bispecific construct includes one or more aminoacid substitutions that promote the heterodimerization of the first andsecond Fc domain monomers. Methods of promoting heterodimerization of Fcdomain monomers are described in detail further herein, see, e.g.,knob-into-hole strategy and electrostatic steering strategy.

In some embodiments, the Fc domain of the bispecific construct ismutated to lack one or more effector functions, typical of a “dead Fcdomain.” In some embodiments, the Fc domain of the bispecific constructis from an IgG1 antibody and includes amino acid substitutions L14A,L15A, and G17A, relative to the sequence of SEQ ID NO: 161 (Table 14) toreduce the interaction or binding between the Fc domain and an Fcγreceptor. In some embodiments, an Fc domain monomer is from an IgG1antibody and includes one or more of amino acid substitutions L234A,L235A, G237A, and N297A (as designated according to the EU numberingsystem per Kabat et al., 1991. In some embodiments, the Fc variantsdescribed herein are minimally glycosylated or have reducedglycosylation. In some embodiments, deglycosylation is accomplished witha mutation of N297A, or by mutating N297 to any amino acid which is notN (as designated according to the EU numbering system per Kabat, et al.(1991)). In some embodiments, the bispecific construct is designed suchthat it has preferential binding to proteins (e.g., receptors such as Fcreceptors) expressed by different cell types. Studies have demonstratedthat amino acid substitutions in the hinge, constant domains (e.g., CH2and CH3 constant domains), or hinge and constant domains of an antibodycan efficiently alter the binding affinities of the antibody towardsspecific receptors (e.g., Fc receptors) expressed on different types ofcells (e.g., regulatory T-cells and effector T-cells). IgG2 having aminoacid substitutions A111S and P112S (relative to SEQ ID NO: 162, Table14) display significantly reduced binding to FcγRIIIa 131 H compared towild-type IgG2. In some embodiments, the Fc variants herein areminimally glycosylated or have reduced glycosylation. In someembodiments, deglycosylation is accomplished with a mutation of N297A,or by mutating N297 to any amino acid which is not N (as designatedaccording to the EU numbering system per Kabat, et al. (1991)). In someembodiments, a bispecific construct includes an Fc domain of the IgG2 orIgG4 subclass. In some embodiments, a bispecific construct including anFc domain of an IgG2 subclass includes amino acid substitutions A111Sand P112S, relative to SEQ ID NO: 162 (Table 14). In some embodiments,the Fc variant comprises a human IgG2 Fc sequence comprising one or moreof A330S, P331S and N297A amino acid substitutions (as designatedaccording to the EU numbering system per Kabat, et al. (1991)).

TABLE 14 IgG Amino Acid Sequences SEQ ID NO: Amino Acid Sequence 161DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G 162ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK

An example of a SIRP-α construct comprising a SIRP-α D1 domain orvariant thereof joined to a first Fc domain monomer by way of a linkerand a second Fc domain monomer, in which the first and second Fc domainmonomers combine to form an Fc domain is shown in FIG. 1. In someembodiments, there is no protein or antibody variable domain attached tothe second Fc monomer. In some embodiments, a SIRP-α construct includesa SIRP-α D1 domain or variant thereof joined to a first Fc domainmonomer by way of a linker and an antibody variable domain joined to asecond Fc domain monomer by way of a linker, in which the first andsecond Fc domain monomers combine to form an Fc domain (as shown in FIG.2). In some embodiments, a SIRP-α construct includes a SIRP-α D1 domainor variant thereof joined to a first Fc domain monomer by way of alinker and a therapeutic protein (e.g., a cytokine, an interleukin, anantigen, a steroid, an anti-inflammatory agent, or an immunomodulatoryagent) joined to a second Fc domain monomer by way of a linker, in whichthe first and second Fc domain monomers combine to form an Fc domain (asshown in FIG. 3). In some embodiments, each of the two Fc domainmonomers in the Fc domain of the SIRP-α constructs described previously(e.g., the SIRP-α constructs as shown in FIGS. 1-3), include amino acidsubstitutions that promote the heterodimerization of the two monomers.Different strategies (e.g., knob-into-hole strategy, electrostaticsteering strategy) and Fc domain amino acid substitutions that promotethe heterodimerization of two Fc domain monomers are described in detailherein. For example, FIG. 4A illustrates a SIRP-α construct having aSIRP-α D1 domain or variant thereof joined to an Fc domain monomerincluding a knob mutation, e.g., T366W, to limit unwanted knob-knobhomodimer formation. FIG. 4B illustrates a SIRP-α construct having ahaving a SIRP-α D1 domain or variant thereof joined to an Fc domainmonomer including hole mutations, e.g., T366S, L358A, and Y407V. In someembodiments, similar Fc domain heterodimerization strategies are appliedto the Fc domains in the constructs described in FIGS. 2 and 3. In someembodiments, a SIRP-α construct includes a fusion protein of a SIRP-α D1domain or variant thereof joined to an Fc domain monomer (as shown inFIG. 5A). In some embodiments, this fusion protein forms a homodimer (asshown in FIG. 5B).

Fc variants of the disclosure coupled with a fusion partner preferablyexhibit reduced or ablated binding to at least one of Fcγ receptorsCD16a, CD32a, CD32b, CD32c, and CD64 as compared to a similarpolypeptide construct comprising the native or wild-type (non-mutated)antibody Fc region. In some cases, the Fc variant or fusion partnerdescribed herein exhibits reduced or ablated binding to the CD16a,CD32a, CD32b, CD32c, and CD64 Fcγ receptors.

In some embodiments, Fc variants of the disclosure coupled with a fusionpartner exhibit reduced binding to complement component C1q and CDCcompared to a similar polypeptide construct comprising the native orwild-type (non-mutated) Fc region. In some cases, the Fc variantexhibits at least a 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%or greater reduction in C1q binding compared to a polypeptide constructcomprising a wild-type Fc region. In some cases, the Fc variant exhibitsreduced CDC compared to a polypeptide construct comprising the native orwild-type (non-mutated) Fc region. In some embodiments, the Fc variantexhibits at least a 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%or greater reduction in CDC compared to a polypeptide constructcomprising a wild-type Fc region.

VIII. Linkers

Disclosed herein, in some embodiments, are polypeptides comprising asignal-regulatory protein α (SIRP-α) D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92.

Also disclosed herein, in some embodiments, are polypeptides comprisingan Fc variant, wherein the Fc variant comprises an Fc domain dimerhaving two Fc domain monomers, wherein each Fc domain monomerindependently is selected from (i) a human IgG1 Fc region consisting ofmutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc regionconsisting of mutations A330S, P331S and N297A; or (iii) a human IgG4 Fcregion comprising mutations S228P, E233P, F234V, L235A, delG236, andN297A.

In the present disclosure, a linker is used to describe a linkage orconnection between polypeptides or protein domains or associatednon-protein moieties. In some embodiments, a linker is a linkage orconnection between an Fc domain monomer, an albumin-binding peptide, oran HSA, and a high affinity SIRP-α D1 variant. In some embodiments, thelinker connects the C-terminus of the SIRP-α D1 variant and theN-terminus of the Fc domain monomer, the albumin-binding peptide, or theHSA, such that the two polypeptides are joined to each other in tandemseries.

In some embodiments, a linker is a simple covalent bond, e.g., a peptidebond, a synthetic polymer such as a polyethylene glycol (PEG) polymer,or any kind of bond created from a chemical reaction, e.g. chemicalconjugation. When a linker is a peptide bond, in some embodiments, thecarboxylic acid group at the C-terminus of one protein domain reactswith the amino group at the N-terminus of another protein domain in acondensation reaction to form a peptide bond. In some embodiments, thepeptide bond is formed from synthetic means through a conventionalorganic chemistry reaction, or by natural production from a host cell,wherein a nucleic acid molecule encoding the DNA sequences of bothproteins (e.g., an Fc domain monomer and a high affinity SIRP-α D1variant) in tandem series can be directly transcribed and translatedinto a contiguous polypeptide encoding both proteins by the necessarymolecular machineries (e.g., DNA polymerase and ribosome) in the hostcell.

When a linker is a synthetic polymer (e.g., a PEG polymer), in someembodiments, the polymer is functionalized with reactive chemicalfunctional groups at each end to react with the terminal amino acids atthe connecting ends of two proteins.

When a linker (except peptide bond mentioned above) is made from achemical reaction, in some embodiments, chemical functional groups(e.g., amine, carboxylic acid, ester, azide, or other functionalgroups), are attached synthetically to the C-terminus of one protein andthe N-terminus of another protein, respectively. In some embodiments,the two functional groups then react through synthetic chemistry meansto form a chemical bond, thus connecting the two proteins together.

Spacers

In the present disclosure, in some embodiments, a linker between an Fcdomain monomer, an albumin-binding peptide, or an HSA, and a polypeptideof the disclosure, is an amino acid spacer including about 1-200 aminoacids. Suitable peptide spacers include peptide linkers containingflexible amino acid residues such as glycine and serine. Examples oflinker sequences are provided in Table 15. In some embodiments, a spacercontains motifs, e.g., multiple or repeating motifs, of GS, GG, GGS,GGG, GGGGS (SEQ ID NO: 163), GGSG (SEQ ID NO: 164), or SGGG (SEQ ID NO:165). In some embodiments, a spacer contains 2 to 12 amino acidsincluding motifs of GS, e.g., GS, GSGS (SEQ ID NO: 166), GSGSGS (SEQ IDNO: 167), GSGSGSGS (SEQ ID NO: 168), GSGSGSGSGS (SEQ ID NO: 169), orGSGSGSGSGSGS (SEQ ID NO: 170). In some embodiments, a spacer contains 3to 12 amino acids including motifs of GGS, e.g., GGS, GGSGGS (SEQ ID NO:171), GGSGGSGGS (SEQ ID NO: 172), and GGSGGSGGSGGS (SEQ ID NO: 173). Insome embodiments, a spacer contains 4 to 12 amino acids including motifsof GGSG (SEQ ID NO: 164), e.g., GGSG (SEQ ID NO: 164), GGSGGGSG (SEQ IDNO: 174), or GGSGGGSGGGSG (SEQ ID NO: 175). In some embodiments, aspacer contains motifs of GGGGS (SEQ ID NO: 163), e.g., GGGGSGGGGSGGGGS(SEQ ID NO: 176). In some embodiments, a spacer contains amino acidsother than glycine and serine, e.g., AAS (SEQ ID NO: 177), AAAL (SEQ IDNO: 178), AAAK (SEQ ID NO: 179), AAAR (SEQ ID NO: 180), EGKSSGSGSESKST(SEQ ID NO: 181), GSAGSAAGSGEF (SEQ ID NO: 182), AEAAAKEAAAKA (SEQ IDNO: 183), KESGSVSSEQLAQFRSLD (SEQ ID NO: 184), GGGGAGGGG (SEQ ID NO:185), GENLYFQSGG (SEQ ID NO: 186), SACYCELS (SEQ ID NO: 187), RSIAT (SEQID NO: 188), RPACKIPNDLKQKVMNH (SEQ ID NO: 189),GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 190), AAANSSIDLISVPVDSR(SEQ ID NO: 191), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO:192).

In some embodiments, a spacer contains motifs, e.g., multiple orrepeating motifs, of EAAAK (SEQ ID NO: 193). In some embodiments, aspacer contains motifs, e.g., multiple or repeating motifs, ofproline-rich sequences such as (XP)n, in which X is any amino acid(e.g., A, K, or E) and n is from 1-5, and PAPAP (SEQ ID NO: 194).

TABLE 15 Linker Sequences SEQ ID NO: Amino Acid Sequence 163 GGGGS 164GGSG 165 SGGG 166 GSGS 167 GSGSGS 168 GSGSGSGS 169 GSGSGSGSGS 170GSGSGSGSGSGS 171 GGSGGS 172 GGSGGSGGS 173 GGSGGSGGSGGS 174 GGSGGGSG 175GGSGGGSGGGSG 176 GGGGSGGGGSGGGGS 177 AAS 178 AAAL 179 AAAK 180 AAAR 181EGKSSGSGSESKST 182 GSAGSAAGSGEF 183 AEAAAKEAAAKA 184 KESGSVSSEQLAQFRSLD185 GGGGAGGGG 186 GENLYFQSGG 187 SACYCELS 188 RSIAT 189RPACKIPNDLKQKVMNH 190 GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG 191AAANSSIDLISVPVDSR 192 GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS 193 EAAAK 194PAPAP

In some embodiments, the length of the peptide spacer and the aminoacids used is adjusted depending on the two proteins involved and thedegree of flexibility desired in the final protein fusion polypeptide.In some embodiments, the length of the spacer is adjusted to ensureproper protein folding and avoid aggregate formation. In someembodiments, a spacer such as a spacer between an HSA and a polypeptidedisclosed herein, is A or AAAL (SEQ ID NO: 178).

IX. Vectors, Host Cells, and Protein Production

Disclosed herein, in some embodiments, are polypeptides comprising asignal-regulatory protein α (SIRP-α) D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92.

Also disclosed herein, in some embodiments, are polypeptides comprisingan Fc variant, wherein the Fc variant comprises an Fc domain dimerhaving two Fc domain monomers, wherein each Fc domain monomerindependently is selected from (i) a human IgG1 Fc region consisting ofmutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc regionconsisting of mutations A330S, P331S and N297A; or (iii) a human IgG4 Fcregion comprising mutations S228P, E233P, F234V, L235A, delG236, andN297A.

In some embodiments, the polypeptides of the disclosure are producedfrom a host cell. A host cell refers to a vehicle that includes thenecessary cellular components, e.g., organelles, needed to express thepolypeptides and fusion polypeptides described herein from theircorresponding nucleic acids. In some embodiments, the nucleic acids areincluded in nucleic acid vectors introduced into the host cell bytransformation, transfection, electroporation, calcium phosphateprecipitation, direct microinjection, infection, etc. In someembodiments, the choice of nucleic acid vectors depend on the host cellto be used. In some embodiments, host cells are of either prokaryotic(e.g., bacterial) or eukaryotic (e.g., mammalian) origin.

In some embodiments, a polypeptide, for example a polypeptide constructcomprising a SIRP-α D1 variant (e.g., any variant provided in Tables 2,5, and 6) and a fusion partner such as an Fc variant, HSA, and analbumin binding peptide, are produced by culturing a host celltransformed with a nucleic acid, preferably an expression vector,containing a nucleic acid encoding the polypeptide construct (e.g., Fcvariant, linker, and fusion partner) under the appropriate conditions toinduce or cause expression of the polypeptide construct. In someembodiments, the conditions appropriate for expression varies with theexpression vector and the host cell chosen. In some embodiments, a widevariety of appropriate host cells are used, including, but not limitedto, mammalian cells, bacteria, insect cells, and yeast. For example, avariety of cell lines that find use in the present disclosure aredescribed in the ATCC® cell line catalog, available from the AmericanType Culture Collection. In some embodiments, Fc variants of thisdisclosure are expressed in a cell that is optimized not to glycosylateproteins that are expressed by such cell, either by genetic engineeringof the cell line or modifications of cell culture conditions such asaddition of kifunensine or by using a naturally non-glycosylating hostsuch as a prokaryote (E. coli, etc.), and in some cases, modification ofthe glycosylation sequence in the Fc is not be needed.

Nucleic Acid Vector Construction and Host Cells

A nucleic acid sequence encoding the amino acid sequence of apolypeptide of the disclosure can be prepared by a variety of methods.These methods include, but are not limited to, oligonucleotide-mediated(or site-directed) mutagenesis and PCR mutagenesis. In some embodiments,a nucleic acid molecule encoding a polypeptide of the disclosure isobtained using standard techniques, e.g., gene synthesis. Alternatively,a nucleic acid molecule encoding a wild-type SIRP-α D1 domain is mutatedto include specific amino acid substitutions using standard techniques,e.g., QuikChange™ mutagenesis. In some cases, nucleic acid molecules aresynthesized using a nucleotide synthesizer or PCR techniques.

In some embodiments, the nucleic acids that encode a polypeptideconstruct, for example a polypeptide construct comprising a SIRP-α D1variant (e.g., any variant provided in Tables 2, 5, and 6) and a fusionpartner such as an Fc variant, HSA, and an albumin binding peptide, areincorporated into an expression vector in order to express the protein.A variety of expression vectors can be utilized for protein expression.Expression vectors can comprise self-replicating, extra-chromosomalvectors or vectors which integrate into a host genome. A vector can alsoinclude various components or elements. For example, in someembodiments, the vector components include, but are not limited to,transcriptional and translational regulatory sequences such as apromoter sequence, a ribosomal binding site, a signal sequence,transcriptional start and stop sequences, translational start and stopsequences, 3′ and 5′ untranslated regions (UTRs), and enhancer oractivator sequences; an origin of replication; a selection marker gene;and the nucleic acid sequence encoding the polypeptide of interest, anda transcription termination sequence. In some embodiments, expressionvectors comprise a protein operably linked with control or regulatorysequences, selectable markers, any fusion partners, additional elements,or any combinations thereof. The term “operably linked” means that thenucleic acid is placed into a functional relationship with anothernucleic acid sequence. Generally, these expression vectors includetranscriptional and translational regulatory nucleic acid operablylinked to the nucleic acid encoding the Fc variant, and are typicallyappropriate to the host cell used to express the protein. A selectiongene or marker, such as, but not limited to, an antibiotic resistancegene or fluorescent protein gene, can be used to select for host cellscontaining the expression vector, for example by antibiotic orfluorescence expression. Various selection genes are available.

In some embodiments, the components or elements of a vector areoptimized such that expression vectors are compatible with the host celltype. Expression vectors which find use in the present disclosureinclude, but are not limited to, those which enable protein expressionin mammalian cells, bacteria, insect cells, yeast, and in in vitrosystems.

In some embodiments, mammalian cells are used as host cells to producepolypeptides of the disclosure. Examples of mammalian cell typesinclude, but are not limited to, human embryonic kidney (HEK) (e.g.,HEK293, HEK293F), Chinese hamster ovary (CHO), HeLa, COS, PC3, Vero,MC3T3, NS0, Sp2/0, VERY, BHK, MDCK, W138, BT483, Hs578T, HTB2, BT20,T47D, NS0 (a murine myeloma cell line that does not endogenously produceany immunoglobulin chains), CRL7030, and HsS78Bst cells. In someembodiments, E. coli cells are used as host cells to producepolypeptides of the disclosure. Examples of E. coli strains include, butare not limited to, E. coli 294 (ATCC® 31,446), E. coli λ 1776 (ATCC®31,537, E. coli BL21 (DE3) (ATCC® BAA-1025), and E. coli RV308 (ATCC®31,608).

Different host cells have characteristic and specific mechanisms for theposttranslational processing and modification of protein products (e.g.,glycosylation). In some embodiments, appropriate cell lines or hostsystems are chosen to ensure the correct modification and processing ofthe polypeptide expressed. Once the vectors are introduced into hostcells for protein production, host cells are cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

In some embodiments, a polypeptide construct, for example a polypeptideconstruct comprising a SIRP-α D1 variant (e.g., any variant provided inTables 2, 5, and 6) and a fusion partner such as an Fc variant, HSA, andan albumin binding peptide, are expressed in mammalian expressionsystems, including systems in which the expression constructs areintroduced into the mammalian cells using virus such as retrovirus oradenovirus. In some embodiments, human, mouse, rat, hamster, or primatecells are utilized. Suitable cells also include known research cells,including but not limited to Jurkat T cells, NIH3T3, CHO, COS, and 293cells. Alternately, in some embodiments, proteins are expressed inbacterial cells. Bacterial expression systems are well known in the art,and include Escherichia coli (E. coli), Bacillus subtilis, Streptococcuscremoris, and Streptococcus lividans. In some cases, polypeptideconstructs comprising Fc variants are produced in insect cells such asbut not limited to Sf9 and Sf21 cells or yeast cells such as but notlimited to organisms from the genera Saccharomyces, Pichia,Kluyveromyces, Hansenula and Yarrowia. In some cases, polypeptideconstructs comprising Fc variants are expressed in vitro using cell freetranslation systems. In vitro translation systems derived from bothprokaryotic (e.g., E. coli) and eukaryotic (e.g., wheat germ, rabbitreticulocytes) cells are available and, in some embodiments, chosenbased on the expression levels and functional properties of the proteinof interest. For example, as appreciated by those skilled in the art, invitro translation is required for some display technologies, for exampleribosome display. In addition, in some embodiments, the Fc variants areproduced by chemical synthesis methods such as, but not limited to,liquid-phase peptide synthesis and solid-phase peptide synthesis. In thecase of in vitro transcription using a non-glycosylating system such asbacterial extracts, the Fc will not be glycosylated even in presence ofthe natural glycosylation site and therefore inactivation of the Fc willbe equivalently obtained.

In some embodiments, a polypeptide construct includes non-natural aminoacids, amino acid analogues, amino acid mimetics, or any combinationsthereof that function in a manner similar to the naturally occurringamino acids. Naturally encoded amino acids generally refer to the 20common amino acids (alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan,

tyrosine, and valine) and pyrrolysine and selenocysteine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, e.g., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, such as,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. In some embodiments, such analogs have modified R groups(such as, norleucine) or modified peptide backbones, but generallyretain the same basic chemical structure as a naturally occurring aminoacid.

Protein Production, Recovery, and Purification

In some embodiments, host cells used to produce polypeptides of thedisclosure are grown in media suitable for culturing of the selectedhost cells. Examples of suitable media for mammalian host cells includeMinimal Essential Medium (MEM), Dulbecco's Modified Eagle's Medium(DMEM), Expi293™ Expression Medium, DMEM with supplemented fetal bovineserum (FBS), and RPMI-1640. Examples of suitable media for bacterialhost cells include Luria broth (LB) plus necessary supplements, such asa selection agent, e.g., ampicillin. In some embodiments, host cells arecultured at suitable temperatures, such as from about 20° C. to about39° C., e.g., from about 25° C. to about 37° C., preferably 37° C., andC02 levels, such as about 5% to 10%. In some embodiments, the pH of themedium is from about pH 6.8 to pH 7.4, e.g., pH 7.0, depending mainly onthe host organism. If an inducible promoter is used in the expressionvector, protein expression can be induced under conditions suitable forthe activation of the promoter.

In some embodiments, protein recovery involves disrupting the host cell,for example by osmotic shock, sonication, or lysis. Once the cells aredisrupted, cell debris is removed by centrifugation or filtration. Theproteins can then be further purified. In some embodiments, apolypeptide of the disclosure is purified by various methods of proteinpurification, for example, by chromatography (e.g., ion exchangechromatography, affinity chromatography, and size-exclusion columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. For example,in some embodiments, the protein is isolated and purified byappropriately selecting and combining affinity columns such as Protein Acolumn (e.g., POROS Protein A chromatography) with chromatographycolumns (e.g., POROS HS-50 cation exchange chromatography), filtration,ultra-filtration, de-salting and dialysis procedures. In someembodiments, a polypeptide is conjugated to marker sequences, such as apeptide to facilitate purification. An example of a marker amino acidsequence is a hexa-histidine peptide (His6-tag), which can bind to anickel-functionalized agarose affinity column with micromolar affinity.As an alternative, a hemagglutinin “HA” tag, which corresponds to anepitope derived from the influenza hemagglutinin protein can be used.

In some embodiments, polypeptides of the disclosure, for example apolypeptide construct comprising a SIRP-α D1 variant (e.g., any variantprovided in Tables 2, 5, and 6) and a fusion partner such as an Fcvariant, HSA, and an albumin binding peptide, are produced by the cellsof a subject (e.g., a human), e.g., in the context of gene therapy, byadministrating a vector such as a viral vector (e.g., a retroviralvector, adenoviral vector, poxviral vector (e.g., vaccinia viral vector,such as Modified Vaccinia Ankara (MVA)), adeno-associated viral vector,and alphaviral vector) containing a nucleic acid molecule encoding apolypeptide of the disclosure. The vector, once inside a cell of thesubject (e.g., by transformation, transfection, electroporation, calciumphosphate precipitation, direct microinjection, infection, etc) can beused for the expression of a polypeptide disclosed herein. In somecases, the polypeptide is secreted from the cell. In some embodiments,if treatment of a disease or disorder is the desired outcome, no furtheraction is required. In some embodiments, if collection of the protein isdesired, blood is collected from the subject and the protein purifiedfrom the blood by various methods.

X. Pharmaceutical Compositions and Preparations

Disclosed herein, in some embodiments, are polypeptides comprising asignal-regulatory protein α (SIRP-α) D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92.

Also disclosed herein, in some embodiments, are polypeptides comprisingan Fc variant, wherein the Fc variant comprises an Fc domain dimerhaving two Fc domain monomers, wherein each Fc domain monomerindependently is selected from (i) a human IgG1 Fc region consisting ofmutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc regionconsisting of mutations A330S, P331S and N297A; or (iii) a human IgG4 Fcregion comprising mutations S228P, E233P, F234V, L235A, delG236, andN297A.

The disclosure features pharmaceutical compositions that includepolypeptides described herein, such as polypeptides having a highaffinity SIRP-α D1 variant. In some embodiments, a pharmaceuticalcomposition of the disclosure includes a polypeptide of the disclosureas the therapeutic protein. In some embodiments, a pharmaceuticalcomposition of the disclosure including a polypeptide described hereinis used in combination with other agents or compositions (e.g.,therapeutic agents, biologics, small molecules, or any combinationsthereof) in a therapy. In some embodiments, one or more additionaltherapeutically active agents, such as for example a small molecule,chemical compound or a biological compound such as polynucleotides andpolypeptides including, but not limited to, siRNA, short polypeptides,and antibodies with therapeutic activity, are optionally formulated inpharmaceutical compositions of polypeptides described herein. In someembodiments, formulations of polypeptide constructs described herein areprepared for storage by mixing a polypeptide construct described hereinhaving the desired degree of purity with optional, pharmaceuticallyacceptable carriers, excipients or stabilizers in the form oflyophilized formulations or aqueous solutions. In some embodiments, apharmaceutical composition of the disclosure includes a nucleic acidmolecule (DNA or RNA, e.g., mRNA) encoding a polypeptide of thedisclosure, or a vector containing such a nucleic acid molecule.

Acceptable carriers, excipients, or stabilizers in a pharmaceuticalcomposition are preferably nontoxic to recipients at the dosages andconcentrations administered. In some embodiments, acceptable carriers,excipients, and stabilizers include buffers such as phosphate, citrate,HEPES, TAE, and other organic acids; antioxidants such as ascorbic acidand methionine; preservatives (e.g., hexamethonium chloride;octadecyldimethylbenzyl ammonium chloride; benzalkonium chloride,benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabenssuch as methyl or propyl paraben; catechol; resorcinol; cyclohexanol;3-pentanol; and m-cresol); low molecular weight (e.g., less than about10 residues) polypeptides; proteins such as human serum albumin,gelatin, dextran, and immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine, histidine,and lysine; monosaccharides, disaccharides, and other carbohydrates suchas glucose, mannose, sucrose, and sorbitol; chelating agents such asEDTA sugars such as sucrose, mannitol, trehalose or sorbitol; sweetenersand other flavoring agents; fillers such as microcrystalline cellulose,lactose, corn and other starches; binding agents; additives; coloringagents; salt-forming counter-ions such as sodium; metal complexes (e.g.,Zn-protein complexes); non-ionic surfactants such as TWEEN™ PLURONICS,™and polyethylene glycol (PEG); or any combinations thereof.

In some embodiments, pharmaceutical compositions that comprisepolypeptides described herein are in a water-soluble form, such as beingpresent as pharmaceutically acceptable salts, which is meant to includeboth acid and base addition salts. The term “pharmaceutically acceptableacid addition salt” refers to those salts that retain the biologicaleffectiveness of the free bases and that are not otherwise undesirable,formed with inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid and the like, and organicacids such as acetic acid, propionic acid, glycolic acid, pyruvic acid,oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid and the like. The term “pharmaceutically acceptable baseaddition salts” includes those derived from inorganic bases such assodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc,copper, manganese, aluminum salts and the like. Particularly preferredare the ammonium, potassium, sodium, calcium, and magnesium salts. Saltsderived from pharmaceutically acceptable organic non-toxic bases includesalts of primary, secondary, and tertiary amines, substituted aminesincluding naturally occurring substituted amines, cyclic amines andbasic ion exchange resins, such as isopropylamine, trimethylamine,diethylamine, triethylamine, tripropylamine, and ethanolamine. Theformulations to be used for in vivo administration are preferablysterile. This can be accomplished by filtration through sterilefiltration membranes or other methods.

In some embodiments, pharmaceutical compositions of the disclosure areadministered parenterally in the form of an injectable formulation. Insome embodiments, pharmaceutical compositions for injection areformulated using a sterile solution or any pharmaceutically acceptableliquid as a vehicle. Pharmaceutically acceptable vehicles include, butare not limited to, sterile water, physiological saline, and cellculture media (e.g., Dulbecco's Modified Eagle Medium (DMEM), α-ModifiedEagles Medium (α-MEM), and F-12 medium). Various formulation methods areavailable.

In some embodiments, the polypeptides described herein are formulated asimmunoliposomes. A liposome is a small vesicle comprising various typesof lipids, phospholipids or surfactants that is useful for delivery of atherapeutic agent to a mammal. Liposomes containing the antibody or Fcfusion can be prepared by various methods known in the art. In someembodiments, the components of the liposome are arranged in a bilayerformation, similar to the lipid arrangement of biological membranes. Insome embodiments, liposomes are generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). In some embodiments, liposomes areextruded through filters of defined pore size to yield liposomes withthe desired diameter. In some embodiments, a chemotherapeutic agent orother therapeutically active agent is optionally contained within theliposome.

In some embodiments, polypeptide constructs described herein and othertherapeutically active agents are entrapped in microcapsules prepared bymethods including, but not limited to, coacervation techniques,interfacial polymerization (for example using hydroxymethylcellulose orgelatin-microcapsules, or poly-(methylmethacylate) microcapsules),colloidal drug delivery systems (for example, liposomes, albuminmicrospheres, microemulsions, nano-particles and nanocapsules), andmacroemulsions.

In some embodiments, sustained-release preparations are prepared.Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymer, which matrices arein the form of shaped articles, e.g., films, or microcapsules. Examplesof sustained-release matrices include polyesters, hydrogels (for examplepoly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides,copolymers of L-glutamic acid and gamma ethyl-L-glutamate,non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolicacid copolymers such as the LUPRON DEPOT™ (which are injectablemicrospheres composed of lactic acid-glycolic acid copolymer andleuprolide acetate), poly-D-(−)-3-hydroxybutyric acid, and ProLease®(commercially available from Alkermes), which is a microsphere-baseddelivery system composed of the desired bioactive molecule incorporatedinto a matrix of poly-DL-lactide-co-glycolide (PLG). Somesustained-release formulations enable release of molecules over a fewmonths, e.g., one to six months, while other formulations releasepharmaceutical compositions of the disclosure for shorter time periods,e.g., days to weeks.

In some embodiments, the concentration of the polypeptide describedherein in a pharmaceutical formulation varies from about 0.1 to 100weight %. In some cases, the concentration of the polypeptide describedherein is in the range of 0.003 to 1.0 molar. In some cases, theconcentration of the polypeptide in a pharmaceutical formulation variesfrom about 5 mg/mL to about 50 mg/mL (e.g., from about 10 mg/mL to about40 mg/mL or from about 20 mg/mL to about 30 mg/mL). In some embodiments,in order to treat a patient, a therapeutically effective dose of apolypeptide described herein is administered. The term “therapeuticallyeffective dose” refers to a dose that produces the effects for which itis administered. The exact dose will depend on the purpose of thetreatment. In some embodiments, dosages range from 0.01 to 100 mg/kg ofbody weight or greater, for example 0.1, 1, 5, 10, 15, 20, 25, 30, 35,40, 45 or 50 mg/kg of body weight. In some embodiments, adjustments forpolypeptide construct degradation, systemic versus localized delivery,and rate of new protease synthesis, as well as the age, body weight,general health, sex, diet, time of administration, drug interaction andthe severity of the condition is necessary.

In some embodiments, the compositions and formulations described hereinare administered to a subject in need thereof. In some embodiments, suchadministration is carried out in vivo. In some embodiments, suchadministration is carried out ex vivo. In some embodiments,administration of the pharmaceutical composition comprising apolypeptide described herein, is done in a variety of ways, including,but not limited to orally, subcutaneously, intravenously, intranasally,intraotically, transdermally, topically (e.g., gels, salves, lotions,creams, etc.), intraperitoneally, intramuscularly, intrapulmonary (e.g.,AERx® inhalable technology commercially available from Aradigm, orInhance™ pulmonary delivery system commercially available from InhaleTherapeutics), vaginally, parenterally, rectally, or intraocularly. Insome embodiments, the pharmaceutical composition is formulatedaccordingly depending upon the manner of introduction.

In some embodiments, the pharmaceutical composition for gene therapy isin an acceptable diluent, or includes a slow release matrix in which thegene delivery vehicle is imbedded. In some embodiments, vectors used asin vivo gene delivery vehicles include, but are not limited to,retroviral vectors, adenoviral vectors, poxviral vectors (e.g., vacciniaviral vectors, such as Modified Vaccinia Ankara), adeno-associated viralvectors, and alphaviral vectors.

XI. Routes, Dosage, and Administration

Disclosed herein, in some embodiments, are polypeptides comprising asignal-regulatory protein α (SIRP-α) D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92.

Also disclosed herein, in some embodiments, are polypeptides comprisingan Fc variant, wherein the Fc variant comprises an Fc domain dimerhaving two Fc domain monomers, wherein each Fc domain monomerindependently is selected from (i) a human IgG1 Fc region consisting ofmutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc regionconsisting of mutations A330S, P331S and N297A; or (iii) a human IgG4 Fcregion comprising mutations S228P, E233P, F234V, L235A, delG236, andN297A.

In some embodiments, pharmaceutical compositions that includepolypeptides of the disclosure as the therapeutic proteins areformulated for, e.g., intravenous administration, parenteraladministration, subcutaneous administration, intramuscularadministration, intra-arterial administration, intrathecaladministration, or intraperitoneal administration. In some embodiments,the pharmaceutical composition is formulated for, or administered via,oral, nasal, spray, aerosol, rectal, or vaginal administration. Forinjectable formulations, various effective pharmaceutical carriers areavailable.

In some embodiments, a pharmaceutical composition that includes anucleic acid molecule encoding a polypeptide of the disclosure or avector containing such nucleic acid molecule is administered by way ofgene delivery. Various methods of gene delivery are available. In someembodiments, vectors used for in vivo gene delivery and expressioninclude, but are not limited to, retroviral vectors, adenoviral vectors,poxviral vectors (e.g., vaccinia viral vectors, such as ModifiedVaccinia Ankara (MVA)), adeno-associated viral vectors, and alphaviralvectors. In some embodiments, mRNA molecules encoding polypeptides ofthe disclosure are administered directly to a subject.

The dosage of the pharmaceutical compositions of the disclosure dependson factors including the route of administration, the disease to betreated, and physical characteristics, e.g., age, weight, generalhealth, of the subject. In some embodiments, the amount of a polypeptideof the disclosure contained within a single dose is an amount thateffectively prevents, delays, or treats the disease without inducingsignificant toxicity. In some embodiments, a pharmaceutical compositionof the disclosure includes a dosage of a polypeptide of the disclosureranging from 0.01 to 500 mg/kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1,2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300,350, 400, 450, or 500 mg/kg) and, in a more specific embodiment, about0.1 to about 50 mg/kg and, in a more specific embodiment, about 1 toabout 30 mg/kg. In some embodiments, the dosage is adapted by aphysician in accordance with the extent of the disease and differentparameters of the subject.

In some embodiments, toxicity of therapeutic agents and polypeptidesdescribed herein is determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., by determining the LD₅₀(the dose lethal to 50% of the population) or the LD₁₀₀ (the dose lethalto 100% of the population). In some embodiments, the data obtained fromthese cell culture assays and animal studies is used in formulating adosage range that is not toxic for use in human. The dosage of theproteins described herein lies preferably within a range of circulatingconcentrations that include the effective dose with little or notoxicity. In some embodiments, the dosage is varied within this rangedepending upon the dosage form employed and the route of administrationutilized. In some embodiments, the exact formulation, route ofadministration and dosage is chosen by an individual physician in viewof the patient's condition.

In some embodiments, the pharmaceutical compositions are administered ina manner compatible with the dosage formulation and in such amount as istherapeutically effective to result in an improvement or remediation ofsymptoms of a disease or disorder. In some embodiments, thepharmaceutical compositions are administered in a variety of dosageforms, e.g., intravenous dosage forms, subcutaneous dosage forms, andoral dosage forms (e.g., ingestible solutions, drug release capsules).Generally, therapeutic proteins are dosed at 0.1-100 mg/kg, e.g., 1-50mg/kg. In some embodiments, pharmaceutical compositions that include apolypeptide of the disclosure are administered to a subject in needthereof, for example, one or more times (e.g., 1-10 times or more)daily, weekly, monthly, biannually, annually, or as medically necessary.Dosages can be provided in either a single or multiple dosage regimens.In some embodiments, the timing between administrations is decreased asthe medical condition improves or increased as the health of the patientdeclines.

XII. Methods of Treatment

Disclosed herein, in some embodiments, are polypeptides comprising asignal-regulatory protein α (SIRP-α) D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92.

Also disclosed herein, in some embodiments, are polypeptides comprisingan Fc variant, wherein the Fc variant comprises an Fc domain dimerhaving two Fc domain monomers, wherein each Fc domain monomerindependently is selected from (i) a human IgG1 Fc region consisting ofmutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc regionconsisting of mutations A330S, P331S and N297A; or (iii) a human IgG4 Fcregion comprising mutations S228P, E233P, F234V, L235A, delG236, andN297A.

Further disclosed herein, in some embodiments, are methods of treatmentcomprising administering polypeptides comprising a signal-regulatoryprotein α (SIRP-α) D1 variant comprising a SIRP-α D1 domain, or afragment thereof, having an amino acid mutation at residue 80 relativeto a wild-type SIRP-α D1 domain; and at least one additional amino acidmutation relative to a wild-type SIRP-α D1 domain at a residue selectedfrom the group consisting of: residue 6, residue 27, residue 31, residue47, residue 53, residue 54, residue 56, residue 66, and residue 92.

In some embodiments, the disclosure provides pharmaceutical compositionsand methods of treatment that are used to treat patients who aresuffering from diseases and disorders associated with SIRP-α or CD47activity, such as cancers and immunological diseases (e.g., autoimmunediseases and inflammatory diseases). In some embodiments, thepolypeptides described herein are administered to a subject in a methodof increasing phagocytosis of a target cell (e.g., a cancer cell) in thesubject. In some embodiments, the polypeptides are administered to asubject in a method to kill cancer cells in the subject. In someembodiments, the polypeptides are administered to a subject in a methodof eliminating regulatory T-cells in the subject. In some embodiments,the polypeptides described herein are administered to a subject in amethod of increasing hematopoietic stem cell engraftment in the subject,wherein the method includes modulating the interaction between SIRP-αand CD47 in the subject. In some embodiments, the polypeptides describedherein are administered to a subject in a method of altering an immuneresponse (e.g., suppressing the immune response) in the subject. In someembodiments, the foregoing methods are coupled with other methods fortreating a disease. In some embodiments, disclosed herein, are acombination of a polypeptide (e.g., a SIRP-α D1 variant) and a secondtherapeutic agent. In some embodiments, the combination comprises apolypeptide (e.g., a SIRP-α D1 variant) and a second therapeutic agent,wherein the second therapeutic agent is an antibody. In someembodiments, the combination comprises a SIRP-α D1 variant comprising aSIRP-α D1 domain, or a fragment thereof, having an amino acid mutationat residue 80 relative to a wild-type SIRP-α D1 domain; and at least oneadditional amino acid mutation relative to a wild-type SIRP-α D1 domainat a residue selected from the group consisting of: residue 6, residue27, residue 31, residue 47, residue 53, residue 54, residue 56, residue66, and residue 92; and an antibody. In some embodiments, thecombination comprises a polypeptide having a sequence according to anyone of SEQ ID NOs: 78-85, 98-104, 107-113, 116-122, 135-137, or 152-159;and an antibody.

In some embodiments, the foregoing methods are employed with strategiesfor treating a disease wherein administration of a polypeptide is atherapeutic option. Non-limiting examples of the foregoing include useof an antibody or a protein fragment. For example, in some embodiments,an antibody or protein fragment is administered in combination with theFc variant polypeptides disclosed herein. In some embodiments, thepolypeptide constructs disclosed herein are used to improve thephagocytosis of other agents.

Methods of treatment include administering to a subject having a disease(e.g., cancer) (i) a polypeptide including a SIRP-α D1 variant andoptionally (ii) an antibody. In some embodiments, before treating adisease (e.g., cancer) in a subject, the amino acid sequence(s) ofSIRP-α in the subject are determined, for example, from each of the twoalleles encoding the SIRP-α gene. In this method of treatment, the aminoacid sequence(s) of SIRP-α polypeptides in a biological sample from thesubject are first determined. The subject is then administered atherapeutically effective amount of a polypeptide of the disclosure. Insome embodiments, the high affinity SIRP-α D1 variant administered hasthe same amino acid sequence as that of SIRP-α polypeptides in thebiological sample of the subject, except for the introduction of aminoacids changes which increase the affinity of the SIRP-α polypeptide toCD47. The high affinity SIRP-α D1 variant in the polypeptide preferablyhas minimal immunogenicity in the subject after the polypeptide isadministered.

In some embodiments, an antibody is administered in addition to thepolypeptides disclosed herein. In some embodiments, the antibody isco-administered with the polypeptide. In some embodiments, the antibodyis administered simultaneously, for example in a pharmaceuticalcomposition having both the polypeptide and the antibody. Alternatively,the antibody is administered either before or after the administrationof the polypeptide. In some embodiments, the polypeptide and theantibody are administered substantially simultaneously (e.g., within oneweek, 6, 5, 4, 3, 2, 1 days, 12, 6, 3, 2, 1 hours of each other, orsubstantially simultaneously), followed by administering the antibodyalone. In some embodiments, the antibody is administered first, followedby administering of the polypeptide and the antibody substantiallysimultaneously (i.e., within one week, 6, 5, 4, 3, 2, 1 days, 12, 6, 3,2, 1 hours of each other, or substantially simultaneously).

An antibody co-administered or provided in a composition or methoddisclosed herein, refers to an antibody that targets a cell, such as acancer cell or a cell of the immune system, such as a T-cell (e.g., aregulatory T-cell). An antibody can be of any immunoglobulin antibodyisotypes, e.g., IgG, IgE, IgM, IgA, or IgD. In some embodiments, theantibody is a human IgG1 isotype antibody. In some embodiments, theantibody is a human IgG2 isotype antibody. In some embodiments, theantibody is a human IgG4 isotype antibody.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multi-specific antibodies (e.g.,bispecific antibodies), antibody fragments, and antibody-like proteinsso long as they exhibit the desired activity. “Antibody fragments”include a portion of an intact antibody, preferably the antigen bindingor variable region of the intact antibody. Examples of antibodyfragments include Fab, Fab′, F(ab′)2, and Fv fragments, diabodies,linear antibodies, single-chain antibody molecules, and multi-specificantibodies. Monoclonal antibody refers to an antibody obtained from apopulation of substantially homogeneous antibodies, e.g., individualantibodies in the population have the same primary sequence except forpossible naturally occurring mutations that can be present in minoramounts. Monoclonal antibodies can be highly specific and directedagainst a single antigenic site (e.g., an epitope on a cancer antigen).In contrast to polyclonal antibody preparations which typically includedifferent antibodies directed against different epitopes, eachmonoclonal antibody is generally directed against a single epitope onthe antigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogenous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. In some embodiments, an antibody in acomposition of the present disclosure causes antibody-dependent cellularphagocytosis (ADCP) or antibody-dependent cellular cytotoxicity (ADCC).Non-limiting examples of diseases that are treated using such strategiesinclude cancers such hematological cancers, for example leukemias (e.g.,acute myeloid leukemia); immune disorders (e.g., to enhance a subject'simpaired or diminished immune response, or alternately to limit asubject's over-active immune response); and pathogenic infections.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide described herein (e.g., a SIRP-α D1 variant) and anantibody that targets a cancer antigen. In some embodiments, a cancerantigen targeted by an antibody or antibody-like protein are exposedpeptides derived from intracellular tumor-associated antigens (TAAs) incomplex with human leukocyte antigen (HLA) class I molecules on thesurface (also known as MHC/peptide complex). Non-limiting examples ofsuch cancer antigens, e.g. peptides in complex with HLA moleculesexposed on the surface of cancer cells, that are targeted by an antibodyor anti-body-like proteins in a composition of the disclosure include:NY-ESO-1/LAGE1, SSX-2, MAGE family (MAGE-A3), gp100/pmel17,Melan-A/MART-1, gp75/TRP1, tyrosinase, TRP2, CEA, PSA, TAG-72, Immaturelaminin receptor, MOK/RAGE-1, WT-1, Her2/neu, EphA3, SAP-1, BING-4,Ep-CAM, MUC1, PRAME, survivin, Mesothelin, BRCA1/2 (mutated), CDK4,CML66, MART-2, p53 (mutated), Ras (mutated), β-catenin (mutated),TGF-βRII (mutated), HPV E6, E7. Examples of such antibodies include ESK1(WT-1), RL1B (Her2-E75), Pr20 (PRAME), and 3.2G1 (hCGβ).

In some embodiments, the methods disclosed herein comprise administeringa polypeptide comprising a SIRP-α D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92; and an antibody that targets a cancer antigen. In someembodiments, the methods disclosed herein comprise administering apolypeptide comprising a SIRP-α D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92; and an antibody that targets NY-ESO-1/LAGE1, SSX-2, MAGEfamily (MAGE-A3), gp100/pmel17, Melan-A/MART-1, gp75/TRP1, tyrosinase,TRP2, CEA, PSA, TAG-72, Immature laminin receptor, MOK/RAGE-1, WT-1,Her2/neu, EphA3, SAP-1, BING-4, Ep-CAM, MUC1, PRAME, survivin,Mesothelin, BRCA1/2 (mutated), CDK4, CML66, MART-2, p53 (mutated), Ras(mutated), β-catenin (mutated), TGF-βRII (mutated), HPV E6, E7. In someembodiments, the methods disclosed herein comprise administering apolypeptide comprising a SIRP-α D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92; and an antibody, wherein the antibody is ESK1 (WT-1),RL1B (Her2-E75), Pr20 (PRAME), and 3.2G1 (hCGβ).

In some embodiments, the methods disclosed herein comprise administeringa polypeptide having a sequence according to SEQ ID NOs: 78-85, 98-104,107-113, 116-122, 135-137, or 152-159. and an antibody that targets acancer antigen. In some embodiments, the methods disclosed hereincomprise administering a polypeptide having a sequence according to anyone of SEQ ID NOs: 78-85, 98-104, 107-113, 116-122, 135-137, or 152-159;and an antibody that targets NY-ESO-1/LAGE1, SSX-2, MAGE family(MAGE-A3), gp100/pmel17, Melan-A/MART-1, gp75/TRP1, tyrosinase, TRP2,CEA, PSA, TAG-72, Immature laminin receptor, MOK/RAGE-1, WT-1, Her2/neu,EphA3, SAP-1, BING-4, Ep-CAM, MUC1, PRAME, survivin, Mesothelin, BRCA1/2(mutated), CDK4, CML66, MART-2, p53 (mutated), Ras (mutated), β-catenin(mutated), TGF-βRII (mutated), HPV E6, E7. In some embodiments, themethods disclosed herein comprise administering a polypeptide having asequence according to any one of SEQ ID NOs: SEQ ID NOs: 78-85, 98-104,107-113, 116-122, 135-137, or 152-159; and an antibody, wherein theantibody is ESK1 (WT-1), RL1B (Her2-E75), Pr20 (PRAME), and 3.2G1(hCGβ).

In some embodiments, an antibody targets cancer cells, for example, bybinding to proteins expressed by cancer cells. Some proteins areexpressed at higher levels in cancer cells than in non-cancer cells. Forexample, a cancer antigen is a protein that is expressed preferentiallyby cancer cells (e.g., it is expressed at higher levels in cancer cellsthan on non-cancer cells) and in some instances it is expressed solelyby cancer cells. Non-limiting examples of proteins, e.g., proteinsexpressed by cancer cells, that are be targeted by an antibody in acomposition of the disclosure include: 4-1BB, 5T4, AGS-16, ALK1, ANG-2,B7-H3, B7-H4, c-fms, c-Met, CA6, CCR4, CD123, CD19, CD20, CD22, CD27,EpCAM, CD30, CD32b, CD33, CD37, CD38, CD40, CD52, CD70, CD74, CD79b,CD98, CEA, CEACAM5, CLDN18.2, CLDN6, CS1, CTLA-4, CXCR4, DLL-4, EGFR,EGP-1, ENPP3, EphA3, ETBR, FGFR2, fibronectin, FR-alpha, Frizzledreceptor, GCC, GD2, glypican-3, GPNMB, HER-2, HER3, HLA-DR, ICAM-1,IGF-1R, IL-3R, LAG-3, LIV-1, mesothelin, MUC16, MUC1, NaPi2b, Nectin-4,Notch 2, Notch 1, OX40, PD-1, PD-L1, PD-L2, PDGFR-α, PS, PSMA, SLTRK6,STEAP1, TEM1, VEGFR, CD25, CD27L, DKK-1, CSF-1R, or any combinationsthereof. In some embodiments, the polypeptides described herein areadministered in combination with a checkpoint inhibitor, such as anantibody inhibitor of CTLA-4 (e.g., ipilimumab, tremelimumab), PD-1(e.g., nivolumab, Pidilizumab, MK3475 also known as pembrolizumab,BMS936559, and MPDL3280A), and LAG-3 (e.g., BMS986016).

In some embodiments, the methods disclosed herein comprise administeringa polypeptide comprising a SIRP-α D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92; and an antibody that targets a protein expressed a by acancer cell. In some embodiments, the methods disclosed herein compriseadministering a polypeptide comprising a SIRP-α D1 variant comprising aSIRP-α D1 domain, or a fragment thereof, having an amino acid mutationat residue 80 relative to a wild-type SIRP-α D1 domain; and at least oneadditional amino acid mutation relative to a wild-type SIRP-α D1 domainat a residue selected from the group consisting of: residue 6, residue27, residue 31, residue 47, residue 53, residue 54, residue 56, residue66, and residue 92; and an antibody that targets 4-1BB, 5T4, AGS-16,ALK1, ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6, CCR4, CD123, CD19, CD20,CD22, CD27, EpCAM, CD30, CD32b, CD33, CD37, CD38, CD40, CD52, CD70,CD74, CD79b, CD98, CEA, CEACAM5, CLDN18.2, CLDN6, CS1, CTLA-4, CXCR4,DLL-4, EGFR, EGP-1, ENPP3, EphA3, ETBR, FGFR2, fibronectin, FR-alpha,Frizzled receptor, GCC, GD2, glypican-3, GPNMB, HER-2, HER3, HLA-DR,ICAM-1, IGF-1R, IL-3R, LIV-1, mesothelin, MUC16, MUC1, NaPi2b, Nectin-4,Notch 2, Notch 1, OX40, PD-1, PD-L1, PD-L2, PDGFR-α, PS, PSMA, SLTRK6,STEAP1, TEM1, VEGFR, CD25, CD27L, DKK-1, CSF-1R, or any combinationthereof. In some embodiments, the methods disclosed herein compriseadministering a polypeptide comprising a SIRP-α D1 variant comprising aSIRP-α D1 domain, or a fragment thereof, having an amino acid mutationat residue 80 relative to a wild-type SIRP-α D1 domain; and at least oneadditional amino acid mutation relative to a wild-type SIRP-α D1 domainat a residue selected from the group consisting of: residue 6, residue27, residue 31, residue 47, residue 53, residue 54, residue 56, residue66, and residue 92; and an antibody, wherein the antibody is an antibodyinhibitor of CTLA-4 (e.g., ipilimumab, tremelimumab), PD-1 (e.g.,nivolumab, Pidilizumab, MK3475 also known as pembrolizumab, BMS936559,and MPDL3280A), or LAG-3 (e.g., BMS986016).

In some embodiments, the methods disclosed herein comprise administeringa polypeptide having a sequence according to any one of SEQ ID NOs:78-85, 98-104, 107-113, 116-122, 135-137, or 152-159; and an antibodythat targets a protein expressed a by a cancer cell. In someembodiments, the methods disclosed herein comprise administering apolypeptide having a sequence according to any one of SEQ ID NOs: SEQ IDNOs: 78-85, 98-104, 107-113, 116-122, 135-137, or 152-159; and anantibody that targets 4-1BB, 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4,c-fms, c-Met, CA6, CCR4, CD123, CD19, CD20, CD22, CD27, EpCAM, CD30,CD32b, CD33, CD37, CD38, CD40, CD52, CD70, CD74, CD79b, CD98, CEA,CEACAM5, CLDN18.2, CLDN6, CS1, CTLA-4, CXCR4, DLL-4, EGFR, EGP-1, ENPP3,EphA3, ETBR, FGFR2, fibronectin, FR-alpha, Frizzled receptor, GCC, GD2,glypican-3, GPNMB, HER-2, HER3, HLA-DR, ICAM-1, IGF-1R, IL-3R, LAG-3,LIV-1, mesothelin, MUC16, MUC1, NaPi2b, Nectin-4, Notch 2, Notch 1,OX40, PD-1, PD-L1, PD-L2, PDGFR-α, PS, PSMA, SLTRK6, STEAP1, TEM1,VEGFR, CD25, CD27L, DKK-1, CSF-1R, or any combinations thereof. In someembodiments, the methods disclosed herein comprise administering apolypeptide having a sequence according to any one of SEQ ID NOs: SEQ IDNOs: 78-85, 98-104, 107-113, 116-122, 135-137, or 152-159; and anantibody, wherein the antibody is an antibody inhibitor of CTLA-4 (e.g.,ipilimumab, tremelimumab), PD-1 (e.g., nivolumab, pidilizumab, MK3475also known as pembrolizumab, BMS936559, and MPDL3280A), or LAG-3 (e.g.,BMS986016).

In some embodiments, the methods disclosed herein comprise administeringa polypeptide described herein (e.g., a SIRP-α D1 variant) and animmuno-oncology antibody. In some embodiments, antibodies that are usedin compositions of the disclosure include, but are not limited to:cetuximab, necitumumab, pembrolizumab, nivolumab, pidilizumab, MEDI0680,MED16469, atezolizumab, avelumab, durvalumab, MEDI6383, RG7888,ipilimumab, tremelimumab, urelumab, PF-05082566, enoblituzumab,vantictumab, varlilumab, mogamalizumab, SAR650984, daratumumab,trastuzumab, trastuzumab emtansine, pertuzumab, elotuzumab, rituximab,ofatumumab, obinutuzumab, RG7155, FPA008, panitumumab, brentuximabvedotin, MSB0010718C, belimumab, bevacizumab, denosumab, panitumumab,ramucirumab, necitumumab, nivolumab, pembrolizumab, avelumab,atezolizumab, durvalumab, MEDI0680, pidilizumab, or BMS-93659, anti-HER2antibody, anti-CD20 antibody, anti-CD19 antibody, anti-CS1 antibody,anti-CD38 antibody, anti-EGFR antibody, anti-PD1 antibody, anti-RANKLantibody, anti-OX40 antibody, anti-PD-1 antibody, anti-PD-L1 antibody,anti-CD274 antibody, anti-CTLA-4 antibody, anti-CD137 antibody,anti-4-1BB antibody, anti-B7-H3 antibody, anti-FZD7 antibody, anti-CD27antibody, anti-CCR4 antibody, anti-CD38 antibody, anti-CSF1R antibody,anti-CSF antibody, anti-CD30 antibody, anti-BAFF antibody, anti-VEGFantibody, or anti-VEGFR2 antibody. In some embodiments, the methodsdisclosed herein comprise administering a polypeptide comprising aSIRP-α D1 variant comprising a SIRP-α D1 domain, or a fragment thereof,having an amino acid mutation at residue 80 relative to a wild-typeSIRP-α D1 domain; and at least one additional amino acid mutationrelative to a wild-type SIRP-α D1 domain at a residue selected from thegroup consisting of: residue 6, residue 27, residue 31, residue 47,residue 53, residue 54, residue 56, residue 66, and residue 92; and anantibody, wherein the antibody is, an anti-HER2 antibody, anti-CD20antibody, anti-CD19 antibody, anti-CS1 antibody, anti-CD38 antibody,anti-EGFR antibody, anti-PD1 antibody, anti-RANKL antibody, anti-OX40antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD274 antibody,anti-CTLA-4 antibody, anti-CD137 antibody, anti-4-1BB antibody,anti-B7-H3 antibody, anti-FZD7 antibody, anti-CD27 antibody, anti-CCR4antibody, anti-CD38 antibody, anti-CSF1R antibody, anti-CSF antibody,anti-CD30 antibody, anti-BAFF antibody, anti-VEGF antibody, oranti-VEGFR2 antibody. In some embodiments, the methods disclosed hereincomprise administering a polypeptide comprising a SIRP-α D1 variantcomprising a SIRP-α D1 domain, or a fragment thereof, having an aminoacid mutation at residue 80 relative to a wild-type SIRP-α D1 domain;and at least one additional amino acid mutation relative to a wild-typeSIRP-α D1 domain at a residue selected from the group consisting of:residue 6, residue 27, residue 31, residue 47, residue 53, residue 54,residue 56, residue 66, and residue 92; and an antibody, wherein theantibody is cetuximab, necitumumab, pembrolizumab, nivolumab,pidilizumab, MEDI0680, MED16469, atezolizumab, avelumab, durvalumab,MEDI6383, RG7888, ipilimumab, tremelimumab, urelumab, PF-05082566,enoblituzumab, vantictumab, varlilumab, mogamalizumab, SAR650984,daratumumab, trastuzumab, trastuzumab emtansine, pertuzumab, elotuzumab,rituximab, ofatumumab, obinutuzumab, RG7155, FPA008, panitumumab,brentuximab vedotin, MSB0010718C, belimumab, bevacizumab, denosumab,panitumumab, ramucirumab, necitumumab, nivolumab, pembrolizumab,avelumab, atezolizumab, durvalumab, MEDI0680, pidilizumab, or BMS-93659.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide comprising a SIRP-α D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92; and an antibody, wherein the antibody is trastuzumab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide comprising a SIRP-α D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92; and an antibody, wherein the antibody is rituximab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide comprising a SIRP-α D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92; and an antibody, wherein the antibody is cetuximab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide comprising a SIRP-α D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92; and an antibody, wherein the antibody is daratumumab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide comprising a SIRP-α D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92; and an antibody, wherein the antibody is belimumab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide comprising a SIRP-α D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92; and an antibody, wherein the antibody is bevacizumab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide comprising a SIRP-α D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92; and an antibody, wherein the antibody is denosumab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide comprising a SIRP-α D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92; and an antibody, wherein the antibody is pantimumab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide comprising a SIRP-α D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92; and an antibody, wherein the antibody is ramucirumab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide comprising a SIRP-α D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92; and an antibody, wherein the antibody is necitumumab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide comprising a SIRP-α D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92; and an antibody, wherein the antibody is nivolumab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide comprising a SIRP-α D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92; and an antibody, wherein the antibody is pembrolizumab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide comprising a SIRP-α D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92; and an antibody, wherein the antibody is avelumab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide comprising a SIRP-α D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92; and an antibody, wherein the antibody is atezolizumab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide comprising a SIRP-α D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92; and an antibody, wherein the antibody is durvalumab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide comprising a SIRP-α D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92; and an antibody, wherein the antibody is MEDI0680.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide comprising a SIRP-α D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92; and an antibody, wherein the antibody is pidilizumab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide comprising a SIRP-α D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92; and an antibody, wherein the antibody is BMS-93659.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide having a sequence according to any one of SEQ ID NOs: SEQID NOs: 78-85, 98-104, 107-113, 116-122, 135-137, or 152-159; and anantibody, wherein the antibody is, an anti-HER2 antibody, anti-CD20antibody, anti-CD19 antibody, anti-CS1 antibody, anti-CD38 antibody,anti-EGFR antibody, anti-PD1 antibody, anti-RANKL antibody, anti-OX40antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD274 antibody,anti-CTLA-4 antibody, anti-CD137 antibody, anti-4-1BB antibody,anti-B7-H3 antibody, anti-FZD7 antibody, anti-CD27 antibody, anti-CCR4antibody, anti-CD38 antibody, anti-CSF1R antibody, anti-CSF antibody,anti-CD30 antibody, anti-BAFF antibody, anti-VEGF antibody, oranti-VEGFR2 antibody. In some embodiments, the methods disclosed hereincomprise administering a polypeptide having a sequence according to SEQID NOs: SEQ ID NOs: 78-85, 98-104, 107-113, 116-122, 135-137, or152-159; and an antibody, wherein the antibody is cetuximab,necitumumab, pembrolizumab, nivolumab, pidilizumab, MEDI0680, MED16469,atezolizumab, avelumab, durvalumab, MEDI6383, RG7888, ipilimumab,tremelimumab, urelumab, PF-05082566, enoblituzumab, vantictumab,varlilumab, mogamalizumab, SAR650984, daratumumab, trastuzumab,trastuzumab emtansine, pertuzumab, elotuzumab, rituximab, ofatumumab,obinutuzumab, RG7155, FPA008, panitumumab, brentuximab vedotin,MSB0010718C, belimumab, bevacizumab, denosumab, panitumumab,ramucirumab, necitumumab, nivolumab, pembrolizumab, avelumab,atezolizumab, durvalumab, MEDI0680, pidilizumab, or BMS-93659.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide having a sequence according to any one of SEQ ID NOs:78-85, 98-104, 107-113, 116-122, 135-137, or 152-159; and an antibody,wherein the antibody is trastuzumab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide having a sequence according to any one of SEQ ID NOs: 7SEQ ID NOs: 78-85, 98-104, 107-113, 116-122, 135-137, or 152-159; and anantibody, wherein the antibody is rituximab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide having a sequence according to any one of SEQ ID NOs: SEQID NOs: 78-85, 98-104, 107-113, 116-122, 135-137, or 152-159; and anantibody, wherein the antibody is cetuximab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide having a sequence according to any one of SEQ ID NOs: SEQID NOs: 78-85, 98-104, 107-113, 116-122, 135-137, or 152-159; and anantibody, wherein the antibody is daratumumab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide having a sequence according to any one of SEQ ID NOs: SEQID NOs: 78-85, 98-104, 107-113, 116-122, 135-137, or 152-159; and anantibody, wherein the antibody is belimumab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide having a sequence according to any one of SEQ ID NOs: SEQID NOs: 78-85, 98-104, 107-113, 116-122, 135-137, or 152-159; and anantibody, wherein the antibody is bevacizumab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide having a sequence according to any one of SEQ ID NOs: SEQID NOs: 78-85, 98-104, 107-113, 116-122, 135-137, or 152-159; and anantibody, wherein the antibody is denosumab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide having a sequence according to any one of SEQ ID NOs: SEQID NOs: 78-85, 98-104, 107-113, 116-122, 135-137, or 152-159; and anantibody, wherein the antibody is pantimumab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide having a sequence according to any one of SEQ ID NOs: SEQID NOs: 78-85, 98-104, 107-113, 116-122, 135-137, or 152-159; and anantibody, wherein the antibody is ramucirumab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide having a sequence according to any one of SEQ ID NOs: SEQID NOs: 78-85, 98-104, 107-113, 116-122, 135-137, or 152-159; and anantibody, wherein the antibody is necitumumab.

In some embodiments, the methods disclosed herein comprise a polypeptidehaving a sequence according to any one of SEQ ID NOs: SEQ ID NOs: 78-85,98-104, 107-113, 116-122, 135-137, or 152-159; and an antibody, whereinthe antibody is nivolumab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide having a sequence according to any one of SEQ ID NOs: SEQID NOs: 78-85, 98-104, 107-113, 116-122, 135-137, or 152-159; and anantibody, wherein the antibody is pembrolizumab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide having a sequence according to any one of SEQ ID NOs: SEQID NOs: 78-85, 98-104, 107-113, 116-122, 135-137, or 152-159; and anantibody, wherein the antibody is avelumab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide having a sequence according to any one of SEQ ID NOs: SEQID NOs: 78-85, 98-104, 107-113, 116-122, 135-137, or 152-159; and anantibody, wherein the antibody is atezolizumab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide having a sequence according to any one of SEQ ID NOs: SEQID NOs: 78-85, 98-104, 107-113, 116-122, 135-137, or 152-159; and anantibody, wherein the antibody is durvalumab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide having a sequence according to any one of SEQ ID NOs: SEQID NOs: 78-85, 98-104, 107-113, 116-122, 135-137, or 152-159; and anantibody, wherein the antibody is MEDI0680.

In some embodiments, the methods disclosed herein comprise a polypeptidehaving a sequence according to any one of SEQ ID NOs: SEQ ID NOs: 78-85,98-104, 107-113, 116-122, 135-137, or 152-159; and an antibody, whereinthe antibody is pidilizumab.

In some embodiments, the methods disclosed herein comprise administeringa polypeptide having a sequence according to any one of SEQ ID NOs: SEQID NOs: 78-85, 98-104, 107-113, 116-122, 135-137, or 152-159; and anantibody, wherein the antibody is BMS-93659.

In some embodiments, the polypeptides disclosed herein enhance theanti-tumor activity of rituximab. In some embodiments, the polypeptidesdisclosed herein enhance the anti-tumor activity of rituximab in theRaji-NSG xenograft model. In some embodiments, the polypeptidesdisclosed herein enhance rituximab-mediated B-cell depletion innon-human primates (NHP).

In some embodiments, the polypeptides and pharmaceutical compositions ofthe disclosure are used in various cancer therapies. The cancersamenable to treatment according to the disclosure include, but are notlimited to, solid tumor cancer, hematological cancer, acute myeloidleukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acutelymphoblastic leukemia, non-Hodgkin lymphoma, Hodgkin lymphoma, multiplemyeloma, bladder cancer, pancreatic cancer, cervical cancer, endometrialcancer, lung cancer, bronchus cancer, liver cancer, ovarian cancer,colon and rectal cancer, stomach cancer, gastric cancer, gallbladdercancer, gastrointestinal stromal tumor cancer, thyroid cancer, head andneck cancer, oropharyngeal cancer, esophageal cancer, melanoma,non-melanoma skin cancer, Merkel cell carcinoma, virally induced cancer,neuroblastoma, breast cancer, prostate cancer, renal cancer, renal cellcancer, renal pelvis cancer, leukemia, lymphoma, sarcoma, glioma, braintumor, and carcinoma. In some embodiments, cancerous conditions amenableto treatment according to the disclosure include metastatic cancers. Insome embodiments, the cancer amenable to treatment according to thedisclosure is a solid tumor or hematological cancer.

In some embodiments, an antibody targets cells of the immune system,such as T-cells, e.g., regulatory T-cells, by binding to proteinsexpressed by cells of the immune system. In some embodiments, themethods disclosed herein comprise administering a polypeptide describedherein (e.g., a SIRP-α D1 variant) and an antibody that targets cells ofthe immune system. Examples of proteins expressed by cells of the immunesystem include, but are not limited to, 41BB, CD40, CD40L, CD163, CD206,CTLA4, PD1, TIM-3, BTLA, VISTA, LAG-3, CD28, OX40, GITR, CD137, CD27,HVEM, CCR4, CD25, CD103, KIrg1, Nrp1, CD278, Gpr83, TIGIT, CD154, CD160,and PD1H. In some embodiments, an antibody is designed such that it haspreferential binding to proteins (e.g., receptors) expressed by T-cells(e.g., regulatory T-cells) as compared to other cells of the immunesystem. In some embodiments, an antibody in a composition of thedisclosure includes an Fc domain of the IgG1, IgG2 or IgG4 subclass.

In some embodiments, the methods of the disclosure include altering animmune response in a subject. The methods include administering thesubject a polypeptide including a high affinity SIRP-α D1 variant and anantibody, thereby altering the immune response in the subject. In someembodiments, altering the immune response includes suppressing theimmune response.

In some embodiments, the polypeptides and pharmaceutical compositions ofthe disclosure are used in various therapies to treat immunologicaldiseases. Autoimmune diseases and inflammatory diseases amenable totreatment according to the disclosure include, but are not limited to,multiple sclerosis, rheumatoid arthritis, a spondyloarthropathy,systemic lupus erythematosus, an antibody-mediated inflammatory orautoimmune disease, graft versus host disease, sepsis, diabetes,psoriasis, atherosclerosis, Sjogren's syndrome, progressive systemicsclerosis, scleroderma, acute coronary syndrome, ischemic reperfusion,Crohn's Disease, endometriosis, glomerulonephritis, myasthenia gravis,idiopathic pulmonary fibrosis, asthma, acute respiratory distresssyndrome (ARDS), vasculitis, and inflammatory autoimmune myositis.

In some embodiments, delivering a polypeptide to a cell involvescontacting the cell with one or more of the compositions describedherein.

Effective doses for such treatment options vary depending upon manydifferent factors, including means of administration, target site,physiological state of the patient, whether the patient is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. In some embodiments, the patient is ahuman, but nonhuman mammals are also be treated, e.g., companion animalssuch as dogs, cats, horses, etc., laboratory mammals such as rabbits,mice, rats, etc., and the like. In some embodiments, treatment dosagesare titrated to optimize safety and efficacy.

In some embodiments, therapeutic dosage range from about 0.0001 to 100mg/kg, and more usually 0.01 to 30 mg/kg, of the host body weight. Insome embodiments, for example, dosages are 1 mg/kg body weight or 30mg/kg body weight or within the range of 1-30 mg/kg. In someembodiments, an exemplary treatment regime entails administration onceevery week or once every two weeks or once a month or once every 3 to 6months. In some embodiments, therapeutic agents and polypeptideconstructs described herein are administered on multiple occasions. Insome embodiments, intervals between single dosages are weekly, monthlyor yearly. In some embodiments, intervals are also irregular asindicated by measuring blood levels of the therapeutic entity in thepatient. Alternatively, the therapeutic agents or polypeptide constructsdescribed herein are administered as a sustained release formulation, inwhich case less frequent administration is possible. In someembodiments, dosage and frequency varies depending on the half-life ofthe polypeptide in the patient.

In prophylactic applications, in some embodiments, a relatively lowdosage is administered at relatively infrequent intervals over a longperiod of time. In some embodiments, patients continue to receivetreatment for the rest of their lives. In other therapeuticapplications, a relatively high dosage at relatively short intervals isrequired until progression of the disease is reduced or terminated, andpreferably until the patient shows partial or complete amelioration ofsymptoms of disease. Thereafter, in some embodiments, the patent isadministered a prophylactic regime.

As used herein, the terms “treatment”, “treating”, and the like, referto administering an agent, or carrying out a procedure, for the purposesof obtaining an effect. In some embodiments, the effect is prophylacticin terms of completely or partially preventing a disease or symptomthereof. In some embodiments, the effect is therapeutic in terms ofaffecting a partial or complete cure for a disease or symptoms of thedisease.

XIII. Kits

Disclosed herein, in some embodiments, are polypeptides comprising asignal-regulatory protein α (SIRP-α) D1 variant comprising a SIRP-α D1domain, or a fragment thereof, having an amino acid mutation at residue80 relative to a wild-type SIRP-α D1 domain; and at least one additionalamino acid mutation relative to a wild-type SIRP-α D1 domain at aresidue selected from the group consisting of: residue 6, residue 27,residue 31, residue 47, residue 53, residue 54, residue 56, residue 66,and residue 92.

Also disclosed herein, in some embodiments, are polypeptides comprisingan Fc variant, wherein the Fc variant comprises an Fc domain dimerhaving two Fc domain monomers, wherein each Fc domain monomerindependently is selected from (i) a human IgG1 Fc region consisting ofmutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc regionconsisting of mutations A330S, P331S and N297A; or (iii) a human IgG4 Fcregion comprising mutations S228P, E233P, F234V, L235A, delG236, andN297A.

Also provided are kits which include polypeptides described herein andinstructions for use of the same. Optionally, the kits can furtherinclude at least one additional reagent. As a non-limiting example, achemotherapeutic agent or anti-tumor antibody could serve as at leastone additional agent. In some embodiments, kits include a labelindicating the intended use of the contents of the kit. The term labelincludes any writing, or recorded material supplied on or with the kit,or which otherwise accompanies the kit.

In some embodiments, a kit includes (i) a polypeptide including a highaffinity SIRP-α D1 variant; optionally (ii) an antibody; and (iii)instructions for administering (i) and (ii) (if provided) to a subjecthaving a disease. In some embodiments, kits include (i) a polypeptideincluding a high affinity SIRP-α D1 variant; and (ii) instructions foradministering (i) with an antibody, for example, an antibody that is notprovided in the kit, to a subject having a disease. In some embodiments,kits include (i) an antibody; and (ii) instructions for administering(i) with a polypeptide including a high affinity SIRP-α D1 variant to asubject having a disease.

In some embodiments, the kits are used to treat a subject having cancer,such as solid tumor cancer, hematological cancer, acute myeloidleukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acutelymphoblastic leukemia, non-Hodgkin lymphoma, Hodgkin lymphoma, multiplemyeloma, bladder cancer, pancreatic cancer, cervical cancer, endometrialcancer, lung cancer, bronchus cancer, liver cancer, ovarian cancer,colon and rectal cancer, stomach cancer, gastric cancer, gallbladdercancer, gastrointestinal stromal tumor cancer, thyroid cancer, head andneck cancer, oropharyngeal cancer, esophageal cancer, melanoma,non-melanoma skin cancer, Merkel cell carcinoma, virally induced cancer,neuroblastoma, breast cancer, prostate cancer, renal cancer, renal cellcancer, renal pelvis cancer, leukemia, lymphoma, sarcoma, glioma, braintumor, carcinoma, or any combinations thereof. In some embodiments, thekits are used to treat a subject having a solid tumor cancer or ahematological cancer.

In some embodiments, the kits are used to treat a subject havingimmunological diseases. In some embodiments, the immunological diseaseis an autoimmune disease or an inflammatory disease, such as multiplesclerosis, rheumatoid arthritis, a spondyloarthropathy, systemic lupuserythematosus, an antibody-mediated inflammatory or autoimmune disease,graft versus host disease, sepsis, diabetes, psoriasis, atherosclerosis,Sjogren's syndrome, progressive systemic sclerosis, scleroderma, acutecoronary syndrome, ischemic reperfusion, Crohn's Disease, endometriosis,glomerulonephritis, myasthenia gravis, idiopathic pulmonary fibrosis,asthma, acute respiratory distress syndrome (ARDS), vasculitis,inflammatory autoimmune myositis, or any combinations thereof.

EXAMPLES Example 1—SIRP-α D1 Variant Polypeptides GeneratingPolypeptides of the Disclosure

A polypeptide of the disclosure including a high affinity SIRP-α D1variant is generated using conventional molecular cloning and proteinexpression techniques. Possible amino acid substitutions in a SIRP-α D1variant relative to a wild-type SIRP-α D1 domain are listed in Tables 2and 5. A nucleic acid molecule encoding a polypeptide of the disclosureis cloned into a vector optimized for expression in bacterial ormammalian cells using well known molecular biology techniques. Afterinduction of protein expression, cells are collected and the expressedpolypeptides are purified from the cell culture supernatant usingaffinity column chromatography. Purified polypeptides are then analyzedby SDS-PAGE, followed by Coomassie Blue staining to confirm the presenceof protein bands of expected size.

Purified polypeptides are screened for binding to CD47 using availabletechniques in the art, such as phage display, yeast display, surfaceplasmon resonance (SPR), scintillation proximity assays, ELISA, ORIGENimmunoassay (IGEN), fluorescence quenching, fluorescence transfer, orany suitable bioassay. The desired polypeptides bind with higheraffinity to CD47, e.g., human CD47, than a wild-type SIRP-α.

Binding Affinity of SIRP-α D1 Variant Polypeptides

In a series of experiments, polypeptides of wild-type SIRP-α D1 domainsand high affinity SIRP-α D1 variants were generated using conventionalmolecular cloning and protein expression techniques. Binding to humanCD47 was determined using SPR as follows: briefly, binding of human CD47(R and D Systems, catalog number 4670-CD or in-house produced asmonomeric extracellular domain, ECD) to wild-type SIRP-α and SIRP-α D1variant polypeptides variants was analyzed on a Biacore T100 instrument(GE Healthcare) or Proteon XPR36 (Bio-rad, Hercules, Calif.) usingphosphate buffered saline (PBS, pH 7.4) supplemented with 0.01% Tween-20(PBST) as running buffer. 200 to 1000 RU of ligand were immobilized in10 mM sodium acetate buffer (pH 4.5) on a Biacore chip CM4 sensor orProteon GLC chip by standard amine coupling following manufacturerrecommendations. Several concentrations of analyte (or SIRP-α D1 variantpolypeptides), e.g., ranging from at least 0.1× to 10×K_(D) value, wereinjected for two minutes with a flow rate 100 L/min, followed by tenminutes of dissociation time. After each analyte injection, the surfacewas regenerated using a 2:1 mixture of Pierce IgG elution buffer (LifeTechnologies, catalog number 21004) and 4 M NaCl injected for 30seconds. Complete regeneration of the surface was confirmed by baselineanalysis and injecting the same analyte at the beginning and end of theexperiment. All sensorgrams were double-referenced using referencesurface and a buffer injection and fitted to 1:1 Langmuir. The analytewas primarily monomeric, either CD47 ECD or SIRP-α without Fc. Ligand onthe chip can be either monomeric or an Fc fusion. Binding data isprovided in Table 16. All SPR assays were performed at 25° C.

TABLE 16 SIRP-α Variant Polypeptide and Associated K_(D) Values HumanCD47 SEQ ID NO: K_(D) (M) 2  0.5 × 10⁻⁶ 53   4.5 × 10⁻¹⁰ 54  2.7 × 10⁻⁹55   6.2 × 10⁻¹⁰ 56   2.0 × 10⁻¹⁰ 57   3.6 × 10⁻¹⁰ 58   1.6 × 10⁻¹⁰ 59 1.4 × 10⁻⁸ 60   3.8 × 10⁻¹⁰ 61   3.8 × 10⁻¹⁰ 62   1.3 × 10⁻¹⁰ 63   8.9× 10⁻¹¹ 64 5.45 × 10⁻⁹ 65  8.00 × 10⁻¹⁰ 66  4.70 × 10⁻¹⁰ 67  2.06 ×10⁻¹⁰ 68  2.51 × 10⁻¹⁰ 69 2.40 × 10⁻⁹ 71 4.94 × 10⁻⁹ 72  7.38 × 10⁻¹⁰ 73 4.48 × 10⁻¹⁰ 74  2.76 × 10⁻¹⁰ 75 1.33 × 10⁻⁹ 76 7.41 × 10⁻⁹ 77  1.14 ×10⁻¹⁰ 78  1.44 × 10⁻¹¹ 79  2.17 × 10⁻¹⁰ 80  4.72 × 10⁻¹¹ 85  1.19 ×10⁻¹⁰

It has also been determined that having a glutamate or aspartate residueat position 54 improves the binding of SIRP-α D1 variant polypeptides tomouse CD47. As a non-limiting example, the SIRP-α D1 variantpolypeptides identified in Table 17 below demonstrate high affinitybinding to mouse CD47. The binding affinity to human CD47 of severalSIRP-α D1 variant polypeptides was compared to the binding affinity tomouse CD47 using SPR as previously described, with mouse CD47 proteinbeing used in place of human CD47 where appropriate. The results arepresented in Table 18.

TABLE 17 SIRP-α Variant Polypeptide Sequences havingImproved Binding to Mouse CD47 SEQ ID NO: Amino Acid Sequence 195EEELQIIQPDKSVLVAAGETATLRCTMTSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS 196EEELQIIQPDKSVLVAAGETATLRCTITSLKPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS 197EEELQIIQPDKSVLVAAGETATLRCTITSLRPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS 198EEELQIIQPDKSVLVAAGETATLRCTITSLYPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS 199EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQRDGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS 200EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGIPDDVEFKSGAGTELSVRAKPS 201EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGMPDDVEFKSGAGTELSVRAKPS 202EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDVEFKSGAGTELSVRAKPS 203EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSSEPDVEFKSGAGTELSVRAKPS 204EEELQIIQPDKSVLVAAGETATLRCTITSLRPVGPIQWFRGAGPGRELIYNQRDGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS 205EEELQIIQPDKSVLVAAGETATLRCTITSLRPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGIPDDVEFKSGAGTELSVRAKPS 206EEELQIIQPDKSVLVAAGETATLRCTITSLRPVGPIQWFRGAGPGRELIYNQRDGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGIPDDVEFKSGAGTELSVRAKPS 207EEELQIIQPDKSVLVAAGETATLRCTITSLYPVGPIQWFRGAGPGRELIYNQRDGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS 208EEELQIIQPDKSVLVAAGETATLRCTITSLYPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGIPDDVEFKSGAGTELSVRAKPS 209EEELQIIQPDKSVLVAAGETATLRCTITSLYPVGPIQWFRGAGPGRELIYNQRDGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGIPDDVEFKSGAGTELSVRAKPS 210EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQRDGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGIPDDVEFKSGAGTELSVRAKPS

TABLE 18 Binding of SIRP-α Variant Polypeptides to Human and Mouse CD47SEQ ID NO: K_(D) (M) − Human K_(D) (M) − Mouse 96 1.04 × 10⁻¹¹ 3.32 ×10⁻⁸ 97 1.55 × 10⁻⁹  >100 nM 100 2.69 × 10⁻⁹  6.32 × 10⁻⁸ 104 9.19 ×10⁻¹¹ 8.04 × 10⁻⁹ 86 1.44 × 10⁻¹¹ 4.30 × 10⁻⁸ 85 8.23 × 10⁻¹¹ 1.14 ×10⁻⁸ 204 3.49 × 10⁻⁰⁹ 5.21 × 10⁻⁹ 206 5.26 × 10⁻⁰⁹ 3.33 × 10⁻⁹ 209 4.46× 10⁻⁰⁹ 4.11 × 10⁻⁹ 210 6.79 × 10⁻⁰⁹ 6.01 × 10⁻⁹

It has also been determined that the N80A mutation, which can minimizeor abrogate partial glycosylation present in certain SIRP-α D1 variantpolypeptides, confers a functional benefit of increasing the homogeneityassociated with SIRP-α D1 variant polypeptides containing such mutation.When SIRP-α variant polypeptides are expressed in E. coli, noglycosylation of N80 will occur due to lack of glycosylation system inE. coli compared to a mammalian system. Table 19 shows that effectivebinding between a SIRP-α D1 variant polypeptide produced in E. coli andhuman CD47 can still occur, thus demonstrating that deglycosylation doesnot affect the binding affinity with which SIRP-α D1 variants can stillbind to CD47. In addition to the N80A mutation, deglycosylation can beaccomplished by mutating N80 to any amino acid which is not N or bydisrupting the motif N-Xaa1-Xaa2 wherein N=asparagine; Xaa1=any aminoacid except P (proline); Xaa2=T (threonine), S (serine) or C (cysteine),wherein the motif refers to residues 80-82 of a SIRP-α D1 variantpolypeptide. By mutating P83 to valine or other residue which is not P,increased glycosylation at N80 can occur and homogenously glycosylatedSIRP-α D1 variant polypeptides can be generated.

The amino acid P83 can also affect the degree of glycosylation. ChangingP83 to any amino acid can increase the efficiency of glycosylation atN80. A SIRP-α D1 variant having a valine (V) at position 83 (SEQ ID NO:213) was expressed in HEK293FS mammalian cells. The size of theexpressed protein was compared to a SIRP-α D1 variant having thewild-type amino acid residue (e.g., proline, P) at position 83 (SEQ IDNO: 71). Molecular weight analysis of the expressed protein on a proteingel (FIG. 18) shows that the variant having a P83V mutation (SEQ ID NO:213, Lane 2) has a higher molecular weight (e.g., ˜22 kDa) compared tothe variant that is unmutated at position 83 (Lane 1). As shown in FIG.18, when residue 83 is mutated to Val, the SIRP-α variant polypeptideexpressed in a mammalian cell host is primarily a molecule at highermolecular weight (˜22 kDa), indicating efficiency for glycosylation atN80 can be increased.

TABLE 19 Representative Binding Data for SIRP-α Variant PolypeptideSequences having Various Glycosylation Profiles SEQ ID NO: KD (M)Expression system 53   4.5 × 10⁻¹⁰ E. coli 58   1.6 × 10⁻¹⁰ E. coli 60  3.8 × 10⁻¹⁰ E. coli 63   8.9 × 10⁻¹¹ E. coli 55   6.2 × 10⁻¹⁰ E. coli62   1.3 × 10⁻¹⁰ E. coli 57   3.6 × 10⁻¹⁰ E. coli 56   2.0 × 10⁻¹⁰ E.coli 61   3.8 × 10⁻¹⁰ E. coli 54  2.7 × 10⁻⁹ E. coli 59  1.4 × 10⁻⁸ E.coli 2  0.5 × 10⁻⁶ E. coli 53   5.2 × 10⁻¹⁰ mammalian cell 77  1.14 ×10⁻¹⁰ mammalian cell 74  2.76 × 10⁻¹⁰ mammalian cell 73  4.48 × 10⁻¹⁰mammalian cell 72  7.38 × 10⁻¹⁰ mammalian cell 75 1.33 × 10⁻⁹ mammaliancell 71 4.94 × 10⁻⁹ mammalian cell 76 7.41 × 10⁻⁹ mammalian cell

Example 2—Generation of Single Arm and Bispecific SIRP-α Polypeptides

The ability of constructs comprising heterodimers of (i) a SIRP-α-Fcfusion protein and (ii) Fc domain monomer fused to a polypeptide, suchas an antigen binding domain, to bind both CD47 and an antigen, e.g.,EGFR, was determined by SPR as previously described in this example. TheFc fusion proteins for forming heterodimers are provided in Table 20.Three monofunctional (e.g., binding one target) SIRP-α-Fc fusions weretested. These fusion proteins are depicted as A, B, C in FIG. 6A. Afirst monofunctional SIRP-α-Fc fusion (“A”) was a homodimer of SEQ IDNO: 136. Second and third monofunctional SIRP-α-Fc fusions wereheterodimers of (i) a SIRP-α-Fc fusion and (ii) a Fc domain monomerwithout an additional polypeptide fused to it. These were generatedusing the Knob & Hole mutation engineering strategies depicted in FIGS.4A and 4B. One monofunctional SIRP-α-Fc fusion (“B”) was formed fromheterodimerization of SEQ ID NO: 139 (an Fc variant) and SEQ ID NO: 142(a SIRP-α-Fc fusion). Another monofunctional SIRP-α-Fc fusion (“C”) wasformed from heterodimerization of SEQ ID NO: 139 (an Fc variant) and SEQID NO: 138 (a SIRP-α Fc fusion). The bifunctional (e.g., binding twotargets) SIRP-α-Fc fusion (“D”) was formed from heterodimerization ofSEQ ID NO: 127 (a SIRP-α Fc fusion) and SEQ ID NO: 144 (an antigenbinding region of Erbitux linked to an Fc variant). SEQ ID NO: 220represents the light chain of the Erbitux antibody.

TABLE 20 Amino Acid Sequences of Fc Fusion Proteins forForming Heterodimers SEQ ID NO: Amino Acid Sequence 138EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 139DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 142EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 144QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCRKTHTCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 220DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 217EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSEKTHTCPECPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Briefly, CD47 was immobilized on a Proteon GLC chip by amine chemistryas described above. In a first injection, analytes (e.g., A, B, C, D,and Erbitux) were injected at 30 uL/min in PBST for 60 s at 100 nM andbinding to the CD47 surface was determined by SPR. In a second,injection 100 nM EGFR-ECD (epidermal growth factor receptorextracellular domain produced in HEK293 cells) was injected and bindingof EGFR-ECD to the CD47-bound analytes was measured. Erbitux did notbind CD47 on the chip and therefore it was not able to bind EGFR in thesecond injection as shown by the curve labeled “Erbitux” in FIG. 6B andillustrated in FIG. 6A. SIRP-α-Fc fusions (e.g., A, B, and C) did bindCD47 but did not bind EGFR in the second injection as shown by thecurves labeled “A,” “B,” and “C” shown in FIG. 6B and illustrated inFIG. 6A. The monomeric proteins, or proteins with one SIRP-α D1 domain(e.g., B and C) higher resonance units than the dimeric protein (e.g.,A) due to a higher amount of molecules bound to the same CD47 sitesavailable on the chip as shown by the curves labeled “B” and “C”,indicating binding to immobilized CD47 and negligible binding toEGFR-ECD (e.g., monofunctionality). Heterodimeric SIRP-α-Erbitux-Fcbound CD47 on the chip and was also able to bind EGFR-ECD in the secondinjection as shown by the curve labeled “D” in FIG. 6B, indicatingbinding to immobilized CD47 and binding of EGFR-ECD (e.g.,bi-functionality).

Example 3—Testing Polypeptides with High Binding Affinity to CD47 inMice

Genetically engineered mouse models of various cancers, e.g., solidtumor and hematological cancer, are used to test the binding ofpolypeptides of the disclosure to CD47. A polypeptide of the disclosureis injected in a mouse, which is dissected at the later time to detectthe presence of the complex of the polypeptide and CD47. Antibodiesspecific to SIRP-α or CD47 are used in the detection.

Example 4—Testing Polypeptides for Immunogenicity

Polypeptides including a high affinity SIRP-α D1 variant are tested inimmunogenicity assays. The polypeptides are tested both in silico and invitro in T-cell proliferation assays, some of which are commerciallyavailable. Polypeptides which provoke a minimal immunogenicity reactionin an in vitro T-cell proliferation assay and display a greater bindingaffinity to CD47 than does wild-type SIRP-α are selected for furtherdevelopment.

Example 5—Testing Polypeptides for In Vivo Toxicity

Different polypeptides including different high affinity SIRP-α D1variants which display various degrees of increased binding affinitiesto CD47 than does wild-type SIRP-α are injected into an animal cancermodel (e.g., a mouse cancer model) to assay the effect of differentbinding affinities on toxicity in the organism. Non-human primate (NHP)can also be used to test high-affinity SIRP-α D1 variants, as the levelof cross reactivity for non-human primate (NHP) CD47 and mouse CD47 maybe different.

Example 6—Fcγ Receptor Binding of Fc Variants

In addition to their ability to modulate target function, therapeuticmonoclonal antibodies and Fc containing fusion proteins are also capableof eliciting two primary immune effector mechanisms: antibody-dependentcell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).ADCC is mediated by Fc region binding to activating Fcγ receptors andpolypeptide constructs comprising Fc variants described herein weretested for Fcγ receptor binding. As shown in Table 21 below, thepolypeptide constructs demonstrated decreased binding to one or more Fcγreceptors as compared to a corresponding wild-type IgG Fc. With regardto IgG1, the mutations L234A, L235A, G237A, and N297A of an IgG1 Fcresulted in a severe loss of binding to Fcγ receptors CD16a, CD32a,CD32b, CD32c, and CD64 as compared to a wild-type IgG1, or a constructlacking one or more of these mutations. Accordingly, the mutationsL234A, L235A, G237A (e.g., IgG1 AAA), along with aglycosylation or thedeglycosylating mutation N297A results in complete loss of binding tothe Fcγ receptors studied. Since Fcγ receptor binding is known to beimportant to phagocytosis, the mutations L234A, L235A, G237A, and N297Acan result in reduction of phagocytosis of the construct comprising theFc variant.

The following materials and methods were used in this example. Bindingof human Fcγ receptors RI (CD64), RIIA (CD32a), RIIB/C (CD32b/c) andRIIIA (CD16a) (R & D Systems, catalog numbers 1257-FC-050, 1330-CD-050,1875-CD-050 and 4325-FC-050 respectively) to Fc variant constructs wasanalyzed on a ProteOn XPR36 instrument (Bio-Rad, Hercules, Calif.) usingphosphate buffered saline (PBS, pH 7.4) supplemented with 0.01% Tween-20as running buffer. Approximately 400 Resonance Unit (RU) of minimallybiotinylated Fc constructs were immobilized on flow cells of a NLCsensor chip (Bio-rad, Hercules, Calif.) by avidin-neutravidininteraction. Biotinylation was performed according to the manufacturer'sinstructions using Pierce EZ-Link Sulfo-NHS-LC-LC-Biotin and anequimolar ratio of linker:protein. Analytes (hFcγR) were injected in a“one-shot” kinetic mode at nominal concentrations of 0, 61, 185, 555,1666, and 5000 nM. Association and dissociation times were monitored for90s and 600s respectively. After each injection, the surface wasregenerated using a 2:1 v/v mixture of Pierce IgG elution buffer (LifeTechnologies, catalog number 21004) and 4 M NaCl. Complete regenerationof the surface was confirmed by injecting the Fc variants at thebeginning and end of the experiment. Biosensor data weredouble-referenced by subtracting the interspot data (containing noimmobilized protein) from the reaction spot data (immobilized protein)and then subtracting the response of a buffer “blank” analyte injectionfrom that of an analyte injection. Double-referenced data were fit to anequilibrium analysis using a simple binding isotherm. K_(D,app). For Fcmolecules with strong binding to hFcγRI, data were also fit globally toa simple Langmuir model and the K_(D,app) value was calculated from theratio of the apparent kinetic rate constants(K_(D app)=k_(d,app)/k_(a,app))

As shown in Table 21, the mutations A330S, P331S, and N297A of an IgG2Fc region resulted in a severe loss of binding to Fcγ receptors CD16a,CD32a, CD32b, CD32c, and CD64 as compared to a wild-type IgG or aconstruct lacking these mutations. Accordingly, the mutations A330S andP331S along with aglycosylation or the deglycosylating mutation N297Aresulted in complete loss of binding to the Fcγ receptors studied. SinceFcγ receptor binding is known to be important to phagocytosis, themutations A330S, P331 S, and N297A are predicted to result in areduction in phagocytosis of the Fc variant. Binding data for IgG4 andvarious mutations are also provided.

TABLE 21 Binding Data (K_(D)) for Fcγ Receptor Binding to Fc Variants.FC description CD16a CD32a CD32b/c CD64 IgG1  370 nM  400 nM 2000 nM0.004 nM IgG1_AAA — 2300 nM —  8000 nM IgG1_N297A — — —   150 nMIgG1_AAA_N297A — — — — IgG2 —  420 nM —   700 nM IgG2_A330S, P331S — 390 nM —   900 nM IgG2_N297A — — — — IgG2_A330S, P331S, — — — — N297A —IgG4 4100 nM  720 nM  710 nM    1 nM IgG4_S228P 3000 nM  810 nM  850 nM   1 nM IgG4_S228P, L235E 2400 nM 1200 nM 1100 nM   60 nM IgG4_S228P,E233P, — 1600 nM —  2100 nM F234V, L235A, delG236 An absence of bindingis represented by “—”

Example 7—C1q Binding Determination of Fc Variants

Complement-dependent cytotoxicity (CDC) is mediated by complementprotein C1q and activation of the complement cascade. Binding of variousconcentrations of C1q complement to various SIRP-α Fc constructs wasdetermined by enzyme-linked immunosorbent assay (ELISA). SIRP-α Fcfusions were prepared at 5 μg/mL in PBS pH 7.4 and used to coatduplicate wells of Nunc Immulon 4HBX ELISA 96 well plates (using 50μL/well) overnight at 4° C. The following day, plates were washed fivetimes with wash buffer (PBS and 0.05% Tween-20) and incubated with 200μL/well of blocking buffer (PBS and 0.5% BSA) for 1 hour at roomtemperature. Plates were washed five times and incubated for 2 hours atroom temperature with 0, 0.13, 0.41, 1.23, 3.7, 11.1, 33.3, 100 μg/mLC1q in assay buffer (PBS, 0.5% BSA, 0.05% Tween-20, 0.25% CHAPS, 5 mMEDTA, and 0.35% NaCl). Plates were washed and incubated for 1 hour with50 l/well HRP Conjugated sheep-anti-human-C1q at 2.0 μg/mL in assaybuffer. Plates were washed five times and incubated for ˜10 minutes withTMB (1-Step Ultra TMB-ELISA, Thermo Sci. Cat. #34028). Finally, 50μL/well Pierce/Thermo Sci. Stop Solution (0.16M sulfuric acid, cat. #N600) was added and plates were read at 450 nm absorbance with a 570 nmreference. Wells lacking SIRP-α-Fc fusion were run to control fornon-specific binding of C1q or the HRP-conjugated detection antibody tothe plate. Wells lacking C1q were run to control for non-specificbinding of the HRP-conjugated detection antibody to a SIRP-α-Fc fusionor to the plate.

As shown in FIG. 14, both wildtype IgG1 (SEQ ID NO: 123) and wildtypeIgG2 (SEQ ID NO: 126) bound C1q in a dose dependent manner. Conversely,IgG1 variants IgG1_AAA (SEQ ID NO: 124); IgG1_N297A (SEQ ID NO: 125);and IgG1_AAA_N297A (SEQ ID NO: 96) demonstrated significantly reducedand minimally detectable C1q binding activity. Likewise, IgG2 variantsIgG2_A330S, P331S (SEQ ID NO: 127); IgG2_N297A (SEQ ID NO: 128); andIgG2_N297A, A330S, P331S (SEQ ID NO: 129) also demonstratedsignificantly reduced and minimally detectable C1q binding activity.This reduced and minimally detectable C1q binding activity of IgG1 andIgG2 variants were comparable to wildtype IgG4 (SEQ ID NO: 130), whichdoes not to bind C1q.

Example 8—Production of Wild-Type Fc and Fc Variants

Using the methods described herein and in accordance with embodiments ofthe disclosure, the wild-type Fc polypeptides and Fc variants of Table 7have been produced.

Example 9—Production of SIRP-α Variant and Fc Variant Polypeptides

Using the methods described herein and in accordance with embodiments ofthe disclosure, the following SIRP-α D1 variant-Fc variant polypeptideswere produced as shown in Table 22 below. Binding to human CD47 wasdetermined by the methodologies as described in Example 1.

TABLE 22 CD47 Binding Affinity of SIRP-α Variant Fc VariantPolypeptides. SEQ ID NO: K_(D) (M) 96  3.51 × 10⁻¹¹ 97 1.09 × 10⁻⁹ 98 8.73 × 10⁻¹¹ 99  8.95 × 10⁻¹⁰ 100 1.79 × 10⁻⁹ 101  8.90 × 10⁻¹⁰ 102 3.79 × 10⁻¹⁰ 103  2.56 × 10⁻¹⁰ 104  9.19 × 10⁻¹¹ 105  3.16 × 10⁻¹¹ 106 8.11 × 10⁻¹⁰ 107  2.19 × 10⁻¹¹ 108  4.78 × 10⁻¹⁰ 109 2.15 × 10⁻⁹ 110 6.53 × 10⁻¹⁰ 111  3.15 × 10⁻¹⁰ 112  2.22 × 10⁻¹⁰ 113  1.32 × 10⁻¹⁰ 114 3.43 × 10⁻¹¹ 115  4.98 × 10⁻¹⁰ 135 3.46 × 10⁻⁹ 136  1.19 × 10⁻¹⁰

Example 10—Phagocytosis of SIRP-α-Fc Variants

To obtain quantitative measurements of phagocytosis, a phagocytosisassay was utilized in which primary human macrophages and GFP+ orCFSE-labeled tumor cells were co-cultured with Fc variant polypeptideconstructs described herein. The following materials and methods wereemployed:

Culture of Tumor Cell Lines

DLD-1-GFP-Luciferase cells, MM1R, and N87 were maintained in growthmedium comprising RPMI (Gibco) supplemented with 10% heat-inactivatedFetal Bovine Serum (Gibco), 1% penicillin/streptomycin (Gibco), and 1%Glutamax (Gibco). DLD-1-GFP-Luciferase and N87 cells were grown asadherent monolayers and MM1R cells were grown in suspension.

Derivation and Culture of Human Monocyte-Derived Macrophages

Whole blood buffy coats were diluted 1:2 with Phosphate Buffered Saline(PBS, Gibco). Diluted blood was split into two tubes and underlayed with20 ml Ficoll-Paque Plus (GE Healthcare). Tubes were centrifuged for 30minutes at 400×g. Peripheral blood mononuclear cells (PBMCs) werecollected from the interface, washed twice by addition of 40 ml PBS,centrifuged for 10 minutes at 100×g, and resuspended in FACS buffer (PBSwith 0.5% Bovine Serum Albumin (Gibco)). CD14+ monocytes were purifiedby negative selection using the Monocyte Isolation Kit II (MiltenyiBiotec) and LS columns (Miltenyi Biotec) according to the manufacturer'sprotocol. CD14+ monocytes were seeded into 15 cm tissue culture plates(Corning) at 10 million cells per dish in 25 ml differentiation mediumcomprised of IMDM (Gibco) supplemented with 10% human AB serum(Corning), 1% penicillin/streptomycin, and 1% Glutamax. Cells werecultured for seven to ten days.

In Vitro Phagocytosis Assays

DLD-1-GFP-Luciferase and N87 cells were detached from culture plates bywashing twice with 20 ml PBS and incubation in 10 ml TrypLE Select(Gibco) for 10 minutes at 37° C. Cells were removed with a cell scraper(Corning), centrifuged, washed in PBS, and resuspended in IMDM. MM1R andN87 cells were labeled with the Celltrace CFSE Cell Proliferation kit(Thermo Fisher) according to the manufacturer's instructions andresuspended in IMDM. Macrophages were detached from culture plates bywashing twice with 20 ml PBS and incubation in 10 ml TrypLE Select for20 minutes at 37° C. Cells were removed with a cell scraper (Corning),washed in PBS, and resuspended in IMDM.

Phagocytosis assays were assembled in ultra-low attachment U-bottom 96well plates (Corning) containing 100,000 DLD-1 GFP Luciferase, MM1R, orN87 cells, five-fold serial dilutions of SIRP-α-Fc variants from 1000 nMto 64 pM, and cetuximab (Absolute Antibody), daratumumab, or controlantibody of the same isotype (Southern Biotech) at 1 μg/ml. Plates werepreincubated 30 minutes at 37° C. in a humidified incubator with 5percent carbon dioxide, then 50,000 macrophages were added. Plates wereincubated two hours at 37° C. in a humidified incubator with 5 percentcarbon dioxide. Cells were pelleted by centrifugation for five minutesat 400×g and washed in 250 μl FACS buffer. Macrophages were stained onice for 15 minutes in 50 μl FACS buffer containing 10 μl human FcRBlocking Reagent (Miltenyi Biotec), 0.5 μl anti-CD33 BV421 (Biolegend),and 0.5 μl anti-CD206 APC-Cy7 (Biolegend). Cells were washed in 200 μlFACS buffer, washed in 250 μl PBS, and stained on ice for 30 minutes in50 μl Fixable Viability Dye eFluor 506 (ebioscience) diluted 1:1000 inPBS. Cells were washed twice in 250 μl FACS buffer and fixed for 30minutes on ice in 75 μl Cytofix (BD Biosciences). Cells were washed in175 μl FACS buffer and resuspended in 75 μl FACS buffer. Cells wereanalyzed on a FACS Canto II (BD Biosciences), with subsequent dataanalysis by Flowjo 10.7 (Treestar). Dead cells were excluded by gatingon the e506-negative population. Macrophages that had phagocytosed tumorcells were identified as cells positive for CD33, CD206, and GFP orCFSE. Five polypeptide constructs comprising SIRP-α D1 domain variantsfused to respective Fc variants were tested for in vitro phagocytosis:

1) (SEQ ID NO: 105) EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 2) (SEQ ID NO: 127)EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 3) (SEQ ID NO: 96)EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 4) (SEQ ID NO: 124)EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 5) (SEQ ID NO: 134)EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSAAAPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

Results

FIG. 7 shows phagocytosis of DLD-1-GFP-Luciferase tumor cells by humanmonocyte-derived macrophages in the presence of SEQ ID NO: 105 (Fcvariant IgG2_A330S, P331S, N297A) and SEQ ID NO: 127 (Fc variantIgG2_A330S, P331S). In particular, FIG. 7 shows that SEQ ID NO: 105 (Fcvariant IgG2_A330S, P331S, N297A) (in the presence or absence of acontrol antibody IgG1,k) has ablated phagocytosis in the phagocytosisassay as a single agent while it is able to increase cetuximab (CTX)phagocytosis (SEQ ID NO: 105+CTX). In contrast, a polypeptide with Fcvariant IgG2_A330S, P331S (SEQ ID NO: 127+IgG1,k) has measurablephagocytosis activity as a single agent. The percent of macrophages thatphagocytosed tumor cells and are GFP⁺ is indicated on the y-axis (FIG.7). Concentration of CD47 binding sites from the addition of SEQ ID NO:105 and SEQ ID NO: 127 is indicated on the x-axis. DLD-1-GFP-Luciferasecells and macrophages were incubated with the indicated concentrationsof SEQ ID NO: 105, SEQ ID NO: 107 and CTX (1 ug/mL) and control antibody(IgG1, k). Cells were also incubated with PBS plus cetuximab (linelabeled PBS+CTX) or a PBS plus a control antibody of the same isotype(line labeled PBS+IgG1k).

FIG. 8 shows phagocytosis of DLD-1-GFP-Luciferase tumor cells by humanmonocyte-derived macrophages in the presence of SEQ ID NO: 96 (Fcvariant IgG1 L234A, L235A, G237A, N297A and SEQ ID NO: 124 (Fc variantIgG1 L234A, L235A, G237A). In particular, FIG. 8 shows that Fc variantIgG1 L234A, L235A, G237A, N297A (SEQ ID NO: 96) and Fc variant IgG1L234A, L235A, G237A (SEQ ID NO: 124) have ablated phagocytosis in thephagocytosis assay as single agents. These are represented by lineslabelled SEQ ID NO 96+IgG1, k and SEQ ID NO: 124+IgG1,k respectively.Interestingly, both polypeptides SEQ ID NO: 96 and SEQ ID NO: 124increased the phagocytosis of a tumor specific antibody, CTX. As shownin FIG. 8, the percent of macrophages that phagocytosed tumor cells andare GFP+ is indicated on the y-axis. Concentration of CD47 binding sitesfrom addition of SEQ ID NO: 96 and SEQ ID NO: 124 is indicated on thex-axis. DLD-1-GFP-Luciferase cells and macrophages were incubated withCTX at 1 μg/mL and the indicated concentrations of SEQ ID NO: 96 (linelabeled SEQ ID NO: 96+CTX) or SEQ ID NO: 124 (line labeled SEQ ID NO:124+CTX). To identify nonspecific effects of cetuximab uponphagocytosis, cells were incubated with a control antibody of the sameisotype as cetuximab and the indicated concentrations of SEQ ID NO: 96(line labeled SEQ ID NO: 96+IgG1,k) or SEQ ID NO: 124 (line labeled SEQID NO: 124+IgG1,k). Cells were also incubated with PBS plus cetuximab(line labeled PBS+CTX) or a PBS plus a control antibody of the sameisotype (line labeled PBS+IgG1k).

FIG. 9 shows phagocytosis of DLD-1-GFP-Luciferase tumor cells by humanmonocyte-derived macrophages in the presence of SEQ ID NO: 134 (Fcvariant IgG4_S228P). In particular, FIG. 9 shows that the SEQ ID NO: 134construct has considerable phagocytosis activity as a single agent in invitro phagocytosis. As shown in FIG. 9, the percent of macrophages thatphagocytosed tumor cells and are GFP+ is indicated on the y-axis.Concentration of CD47 binding sites from addition of SEQ ID NO: 134 isindicated on the x-axis. DLD-1-GFP-Luciferase cells and macrophages wereincubated with the indicated concentrations of SEQ ID NO: 134 (linelabeled SEQ ID NO: 134+Medium). Cells were also incubated with controlantibody (IgG1, k; black square).

Example 11—Production of SIRP-α Variant and HSA Polypeptides

Additionally, using the methods described herein and in accordance withembodiments of the disclosure, SIRP-α D1 variant polypeptide wasexpressed by fusion to HSA polypeptides, as shown in Table 23 below.Binding to human CD47 was determined by the methodologies as describedin Example 1.

TABLE 23 CD47 Binding Affinity of SIRP-α Variants Fused to HSAPolypeptides SEQ ID NO: K_(D) (M) 150  4.53 × 10⁻¹⁰ 151 5.54 × 10⁻⁹ 152 2.78 × 10⁻¹⁰ 153 4.24 × 10⁻⁹ 154  2.35 × 10⁻¹⁰ 155 1.11 × 10⁻⁸ 157 2.15× 10⁻⁹ 158 1.09 × 10⁻⁹ 159   7.6 × 10⁻¹⁰

Example 12—Extended Half-Life Associated with SIRP-α VariantPolypeptides

As shown in Table 24 and FIG. 10, SIRP-α D1 variant polypeptidescomprising Fc and HSA fusions can have an extended half-life compared toa SIRP-α D1 variant alone. For example, the SIRP-α D1 variantpolypeptide fused to Fc as represented by SEQ ID NO: 104 and the SIRP-αD1 variant polypeptide fused to HSA as represented by SEQ ID NO: 159have increased half-life relative to a SIRP-α D1 variant polypeptidewhich is not fused to an Fc or HSA as represented by SEQ ID NO: 85. Thehalf-life extension can be attributed to the ability of SIRP-α D1variant polypeptides which are fused to Fc and HSA to bind to FcRn,which may be associated with prolonged cycling.

TABLE 24 Half-life Measurements for Single Dose Treatments with SIRP-αVariant Polypeptides Dosage Half-life SEQ ID NO: amount (hour) 104 10mg/kg 41.10 159 10 mg/kg 24.54 85 10 mg/kg 8.20

The methodologies used for this Example are as follows. Briefly, CD-1male mice weighing approximately 25 grams were obtained from HarlanLabs, and were used for the single dose PK study of the compoundsrepresented by SEQ ID NO: 104, SEQ ID NO: 159, and SEQ ID NO: 85. Eachcompound was formulated at a working dose of 5 mg/mL. The volume of thedose was adjusted based on the weight of each mouse, ensuring that eachmouse was dosed at 1, 3 and 10 mg/kg. The compounds were administeredintravenously via the mouse tail vein. Three mice were dosed for eachtime point at each dose level for each compound. After dosing, mice hadblood withdrawn at the following 8 time points: 0.25, 1, 4, 8, 24, 48,72 and 120 hrs. 500 μL of whole blood was collected into microtainertubes by orbital bleed. Whole blood samples were rested for 30 minutesto allow serum separation. Samples were then centrifuged for 10 min at4° C. at a RCF of 1000. Serum was then transferred to 0.5 mL tubeswithin 40 min of processing and kept frozen until analysis.

The data for SEQ ID NO: 104 was obtained using a human Fc ELISAprotocol. Briefly, Immulon 4HBX ELISA 96 well plates were coated (ThermoScientific cat. #3855) with 2 μg/ml, 100 μl/well of purified CD47overnight at room temperature in 1× antigen coating buffer(ImmunoChemistry Technologies, cat. #6248). Wells were washed 5 timeswith 200-300 μL/well 1×TBST (Tris-Buffered Saline+0.05% Tween-20)(Thermo Scientific 20×, cat. #28360). Wells were blocked with 200μL/well 7.5% BSA in PBS (GIBCO, cat. #15260-037) for 1-2 hours. Wellswere washed 5 times with 200-300 μL/well 1×TBST. 50 μL/well standardcurve, Quality Controls (QCs) and unknown samples diluted in normal CD1mouse serum diluted 1:4 in TBS was added. The standard curve, QCs andunknown samples were incubated at room temperature for 1 hour.Concentrations for standard curve were as follows: 0.2500 μg/mL; 0.1250μg/mL; 0.0625 μg/mL; 0.0313 μg/mL; 0.0156 μg/mL; 0.0078 μg/mL; 0.0039μg/mL; 0.0020 μg/mL; 0.0010 μg/mL; 0.0005 μg/mL; 0.00025 μg/mL; and0.00000 μg/mL. Quality Controls (QCs) were frozen and aliquoted, andstandard curve protein at a “high,” “mid,” and “low” concentrations onthe linear curve of the standard curve which served as controls toensure that the assay was working well were as follows: QC High=0.125μg/ml; QC Mid=0.016 μg/ml; and QC Low=0.004 μg/ml.

Then, the wells were washed 5 times with 200-300 μL/well 1×TBST. 50μL/well of 0.25 ug/mL Abbexa Goat anti-Human IgG Fc polyclonal antibody(11.6 mg/mL stock, Abbexa cat. # abx023511) diluted into 1×TBST+1% BSAwas added and incubated for 1 hour at room temperature. Plates werewashed5 times with 200-300 μL/well 1×TBST. 50 μL/well of 0.125 μg/mLZyMax/Invitrogen rabbit anti-goat IgG-HRP conjugated (Thermo Scientific,cat. #81-1620), diluted into TBST+1% BSA was added and incubated for 1hour at room temperature. Wells were washed 6 times with 200-300 μL/well1×TBST. The following steps and reagents were carried out at roomtemperature: 0 μL/well room temperature 1-Step Ultra TMB-ELISA (ThermoScientific cat. #34028) was added and incubated 2-5 minutes at roomtemperature until color development was sufficient. 50 μL/well of roomtemperature Stop Solution (0.16M sulfuric acid, Thermo Scientific cat. #N600) was added immediately and mixed well. Plates were read immediatelyin a spectrophotometer at O.D. 450 and at O.D. 570. The O.D. 570 readingwas a background reading which was subtracted from the O.D. 450 reading.Using a software program like Molecular Devices SoftMax Pro or Graph PadPrism, the standard curve values were plotted using a 4 parameter fitcurve and the concentrations of the unknown samples were interpolatedfrom the standard curve using the software.

The data for SEQ ID NO: 85 was obtained using a His Tag ELISA protocol.Immulon 4HBX ELISA 96 well plates (Thermo Scientific cat. #3855) werecoated with 2 μg/mL, 100 μL/well of purified CD47 overnight at roomtemperature in 1× antigen coating buffer (ImmunoChemistry Technologies,cat. #6248). Wells were washed 5 times with 200-300 μL/well using 1×TBST(Tris-Buffered Saline+0.05% Tween-20) (Thermo Scientific 20×, cat.#28360). Wells were blocked with 200 μL/well 7.5% BSA in PBS (GIBCO,cat. #15260-037) for 1-2 hours. Wells were washed 5 times with 200-300μL/well 1×TBST. 50 μL/well standard curve, Quality Controls (QCs) andunknown samples diluted in normal CD1 mouse serum diluted 1:4 in TBSwere added. The standard curve, QCs and unknown samples were incubatedat room temperature for 1 hour. The standard curve concentrations wereas follows: 0.12500 μg/mL; 0.06250 μg/mL; 0.03125 μg/mL; 0.01563 μg/mL;0.00781 μg/mL; 0.00391 μg/mL; 0.00195 μg/mL; 0.00098 μg/mL; and 0.00000μg/mL. Quality Controls (QCs) were frozen and aliquoted, and standardcurve protein at a “high,” “mid,” and “low” concentration on the linearcurve of the standard curve which served as controls to ensure that theassay was working well were as follows: QC High=0.02 μg/ml; QC Mid=0.01g/ml; and QC Low=0.005 μg/ml.

Thereafter, wells were washed 5 times with 200-300 μL/well 1×TBST. 50μL/well of 0.2 μg/mL Abcam rabbit anti-6×His Tag-HRP conjugatedpolyclonal antibody (1 mg/mL stock, abcam cat. # ab1187) diluted intoTBST+1% BSA was added and incubated for 1 hour at room temperature.Plates were washed 6 times with 200-300 uL/well 1×TBST. Thereafter, thefollowing steps and agents were carried out at room temperature. 50L/well of room temperature 1-Step Ultra TMB-ELISA (Thermo Scientificcat. #34028) was added and incubated 3-5 minutes at room temperatureuntil color development was sufficient. 50 μL/well of room temperatureStop Solution (0.16M sulfuric acid, Thermo Scientific cat. # N600) wasimmediately added and mixed well. Plates were read immediately in aspectrophotometer at O.D. 450 and at O.D. 570. The O.D. 570 reading wasa background reading which was subtracted from the O.D. 450 reading.Using a software program such as Molecular Devices SoftMax Pro or GraphPad Prism, the standard curve values were plotted using a 4 parameterfit curve and the concentrations of the unknown samples wereinterpolated from the standard curve using the software.

The data for SEQ ID NO: 159 was obtained using a HSA ELISA protocol.Immulon 4HBX ELISA 96 well plates (Thermo Scientific cat. #3855) werecoated with 2 ug/ml, 100 ul/well of purified CD47 overnight at roomtemperature in 1× antigen coating buffer (ImmunoChemistry Technologies,cat. #6248). Wells were washed 5 times with 200-300 μL/well using 1×TBST(Tris-Buffered Saline+0.05% Tween-20) (Thermo Scientific 20×, cat.#28360). Wells were blocked with 200 μL/well Li-Cor Odyssey BlockingBuffer (TBS) (Li-Cor, cat. #927-50000) for 2 hours, and blocking bufferscontaining albumin were not used. Wells were washed 5 times with 200-300μL/well 1×TBST. 50 uL/well standard curve, Quality Controls (QCs) andunknown samples diluted in normal CD1 mouse serum diluted 1:4 in TBS wasadded. The standard curve, QCs and unknown samples were incubated atroom temperature for 1 hour.

The standard curve concentrations were as follows: 3.20 μg/ml; 1.60μg/ml; 0.80 μg/ml; 0.40 μg/ml; 0.20 μg/ml; 0.10 μg/ml; 0.05 μg/ml; 0.025μg/ml; and 0.00 μg/ml. Quality Controls (QCs) are frozen and aliquoted,and standard curve protein at a “high”, “mid”, and “low” concentrationson the linear part of the standard curve which served as controls toensure that the assay was working well were as follows: QC High=0.6μg/ml; QC Mid=0.3 μg/ml; QC Low=0.15 μg/ml, and QC Low=0.01 μg/ml.

Thereafter, wells were washed 5 times with 200-300 μL/well 1×TBST. 50μL/well of 1 g/ml Thermo Scientific/Pierce rabbit anti-HSA-HRPconjugated (Thermo Scientific cat. # PA1-26887) diluted into 1×TBST wasadded and incubated for 1 hour at room temperature. Plates were washed 6times with 200-300 L/well 1×TBST. Thereafter, the following steps andreagents were carried out at room temperature. 50 L/well roomtemperature 1-Step Ultra TMB-ELISA substrate (Thermo Scientific, cat.#34028) was added and incubated 3-5 minutes at room temperature untilcolor development was sufficient. 50 μl/well of room temperature TMBStop Solution (0.16 M Sulfuric Acid solution, Thermo Scientific cat. #N600) was added and mixed well. Plates were read immediately in aspectrophotometer at O.D. 450 and at O.D. 570. The O.D. 570 reading wasa background reading which was subtracted from the O.D. 450 reading.Using a software program such as Molecular Devices SoftMax Pro or GraphPad Prism, the standard curve values were plotted using a 4 parameterfit curve and the concentrations of the unknown samples wereinterpolated from the standard curve using the software.

Example 13—Reduced Hemagglutination Demonstrated by SIRP-α VariantPolypeptides

As shown in FIG. 11, SIRP-α D1 variant polypeptides demonstrated reducedor ablated hemagglutination. Specifically, when hemagglutination occurs,a diffused red coloration is present instead of a red dot, as is shownfor the positive control B6H12. For the SIRP-α D1 variant polypeptidestested in FIG. 11, there was a reduction or ablation ofhemagglutination.

The methodologies used for this Example were as follows: human wholeblood buffy coats were received from the Stanford University BloodCenter and diluted 1:2 with Phosphate Buffered Saline (PBS, Gibco).Diluted blood was split into two tubes and underlayed with 20 mlFicoll-Paque Plus (GE Healthcare). Tubes were centrifuged for 30 minutesat 400×g. Supernatants were removed and the erythrocyte pellets werewashed twice by addition of 30 mL of PBS and centrifugation at 3500 RPM.Thereafter, a hemagglutination assay was carried out as follows: humanerythrocytes were diluted in PBS and transferred to 96 well polystyreneplates (Corning) at 4 million cells per well in a volume of 75 μL.Five-fold serial dilutions of the indicated proteins were added to wellsin a volume of 75 μL PBS, with final concentration from 1000 nM to 0.488nM. As a negative control, PBS alone was added to one row of wells.Erythrocytes settled to the well bottom, forming a small andwell-defined pellet. As a positive control, cells were treated with theanti-CD47 antibody B6H12 (ebioscience). This antibody causedhemagglutination at concentrations between 8 and 63 nM, indicated byformation of a large and diffuse cell pellet. Among tested constructs,IgG2-based polypeptides (SEQ ID NO: 109 and SEQ ID NO: 113) causedslight hemagglutination at 4 and 8 nM. No hemagglutination was observedfor all other polypeptide constructs (IgG1-based and HSA-based).

Example 14—Anti-Tumor Activity of SEQ ID NO: 211 in a Mouse SyngeneicTumor Model

C57BL/6 mice (7- to 10-week-old female animals) obtained from CharlesRiver Laboratory were used. Mouse colon adenocarcinoma cell line MC38was recovered from frozen stocks and grown in RPMI 1640 containing 10%fetal bovine serum, penicillin-streptomycin, and L-glutamine. Cells werespun down and resuspended at a concentration of 2E+07 cells/mL inserum-free medium without additives. On Day −7 (i.e., 7 days before theprojected staging day), the mice were implanted by subcutaneousinjection into the left flank with 100 μL (2.0×10⁶ cells) per mouse ofthe freshly prepared MC38 in phosphate buffered saline (PBS). When thetumors reached a mean volume of approximately 50 mm³, fifty animals withestablished tumors and moderate body weights were randomized into 5treatment groups (Group 1-5, n=10 mice each). Starting on Day 1, mice ofGroups 1 to 5 were treated with vehicle (PBS), anti-mPD-L1 (Clone10F.9G2, 200 μg), SEQ ID NO: 211 (200 μg), anti-mPD-L1 (200 μg)+SEQ IDNO: 211 (100 μg), or anti-mPD-L1 (200 μg)+SEQ ID NO: 211 (200 μg),respectively. Doses were by administered intraperitoneal (IP) injectionof 0.05 mL/mouse on days 1, 4 and 7.

SEQ ID NO: 211 was generated by genetically fusing SED ID NO: 206 to aFc domain monomer. SEQ ID NO: 206 contains mutations shown to improvebinding to mouse CD47. The binding data is presented in Table 18.

6) SEQ ID NO: 211 EEELQIIQPDKSVLVAAGETATLRCTITSLRPVGPIQWFRGAGPGRELIYNQRDGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGIPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Clinical observations and body weights were monitored throughout thestudy up to Day 42. Tumor sizes were measured two times per week, and atstudy completion, the perpendicular minor (width, W, and height, H) andmajor (length, L) dimensions were measured using microcalipers(Mitutoyo, Aurora, Ill.). Tumor volume (mm³) was calculated using theformula for the volume of an ellipsoid sphere (L×W×H/2). Study animalswere subjected to humane sacrifice during the study when tumor volumesin individual animals exceeded (or approached) 2,500 mm³. The number ofanimals remaining in the study up to Day 42 were used for a survivalanalysis.

Tumors grew to various degrees in all five groups. Among mice dosed withvehicle or SEQ ID NO: 211 (200 μg) (Groups 1 and 3, respectively),sacrifices began during the 4^(th) week (from Day 25) and all animals inthese groups were dead by the end of the 5^(th) week (Day 35). Amongmice dosed with anti-mPD-L1 (alone or in combination with SEQ ID NO:211; Groups 2, 4, and 5), sacrifices began during the 5^(th) week (fromDay 29 or 32) but a subset (40-70%) of these animals survived to thescheduled study end (Day 42). FIG. 12 shows survival curves for eachtreatment group during the study period. Numerically, the anti-mPD-L1plus 200 μg SEQ ID NO: 211 treatment group had the highest number ofsurviving animals, following by the anti-mPD-L1 plus 100 μg SEQ ID NO:211 treatment group and anti-mPD-L1 alone group, with 7 out 10 (70%), 5out 10 (50%) and 4 out of 10 (40%) mice remaining at Day 42,respectively (Table 25). Median survival was 29 and 30.5 daysrespectively for vehicle (Group 1) and SEQ ID NO: 211 alone (Group 3)treatments. Median survival increased to 42 days for anti-mPD-L1 alone(Group 2) and anti-PD-L1 plus 100 μg SEQ ID NO: 211 (Group 4) treatment.Median survival for anti-mPD-L1 plus 200 μg SEQ ID NO: 211 treatment(Group 5) was not determined as more than 50% of animals remained at theend of the study (Day 42).

TABLE 25 Animal Survival Data. Treatment (3 IP doses - on Number ofAnimals Alive on Indicated Day Group Days 1, 4 and 7) 1 4 7 11 14 18 2225 29 32 35 39 42 1 PBS 10 10 10 10 10 10 10 6 1 0 0 0 0 2 anti-mPD-L1(200 μg) 10 10 10 10 10 10 10 10 9 9 7 5 4 3 SEQ ID NO: 211 (200 μg) 1010 10 10 10 10 10 9 5 1 0 0 0 4 anti-mPD-L1 (200 μg) + 10 10 10 10 10 1010 10 10 9 7 6 5 SEQ ID NO: 211 (100 ug) 5 anti-mPD-L1 (200 ug) + 10 1010 10 10 10 10 10 9 9 8 7 7 SEQ ID NO: 211 (200 μg)

Tumors exhibited rapid growth in the vehicle treated group, indicatingongoing tumor growth in the absence of effective treatment. Dosing with200 μg SEQ ID NO: 211 (Group 3) yielded significant attenuation of tumorgrowth only at intermittent time points (Day 7 and 14, for both raw andnormalized tumor volume) compared to dosing with vehicle. Dosing with200 μg anti-mPD-L1, alone or in combination with SEQ ID NO: 211 (Groups2, 4, and 5), provided significant attenuation of tumor growth from Day4 or 7 (for raw or normalized tumor volume, respectively) compared todosing with vehicle (FIG. 13 and Table 26). The addition of SEQ ID NO:211 to the anti-mPD-L1 regimen (Group 2 vs. 4 or Group 2 vs. 5) producedadditional tumor growth inhibition over anti-mPD-L1 treatment alone.Day-22 tumor volumes, including tumor growth inhibition (% TGI), areprovided in Table 26. Day 22 is used for the comparison because this dayis the last time point at which all animals were still alive. Tumorgrowth inhibition (% TGI) on Day 22 vs Day 1 were 83%, 81% and 77% foranti-mPD-L1 plus 200 μg SEQ ID NO: 211 group, anti-mPD-L1 plus 100 μgSEQ ID NO: 211 group and anti-mPD-L1 alone group, respectively (Table26).

TABLE 26 Tumor Volume Analysis Day-1 Day-22{circumflex over ( )} MeanAgent Tumor Tumor Day 22* vs. Day 1 Normalized (Three doses VolumeVolume T-B (mm³) % TGI Day-22{circumflex over ( )} on Day 1, 4 (B, mm³)(T, mm³) Δ tumor % Group Volume Group and 7, IP) Mean ± SD Mean ± SDvolume 1 Δ % Day 1 1 Vehicle 52 ± 13 2126 ± 599  2074.2  0% 4380% 2anti-mPD-L1 52 ± 12 537 ± 464 484.6 77% 1062% (200 μg) 3 SEQ ID NO: 51 ±12 1697 ± 679  1645.4 21% 3384% 211 (200 μg) 4 anti-mPD-L1 52 ± 13 456 ±368 404.3 81%  959% (200 μg) + SEQ ID NO: 211 (100 μg) 5 anti-mPD-Ll 52± 11 399 ± 497 347.9 83%  728% (200 μg) + SEQ ID NO: 211 (200 μg)0 *Day22 is the last day on which all animals of all groups remained alive.

Example 15—Optimizing Combination Therapy for Treating Cancer

Polypeptides including a high affinity SIRP-α D1 variant areco-administered with checkpoint inhibitors to treat mouse models ofvarious cancers, e.g., solid tumor and hematological cancer. Cancers maybe recognized by the immune system, and under some circumstances, theimmune system may be involved in eliminating tumors. Blockade ofco-inhibitory molecules, such as CTLA-4, PD-1, and LAG-3, may beinvolved in amplifying T-cell responses against tumors. Polypeptidesdescribed herein are administered in combination with a checkpointinhibitor, such as an antibody inhibitor of CTLA-4 (e.g., ipilimumab,tremelimumab), PD-1 (nivolumab, pidilizumab, MK3475 also known aspembrolizumab, BMS936559, and MPDL3280A), and LAG-3 (e.g., BMS986016).

Established A20 tumors in BALB/c mice (e.g., lymphoma models) aretreated with an antibody inhibitor of CTLA-4 and a high affinity SIRP-αD1 variant fused to an IgG Fc variant provided herein (e.g., a SIRP-αconstruct). Starting on Day 1, mice are treated with vehicle (PBS),tremelimumab (200 μg)+SIRP-α construct (100 pg), or tremelimumab (200μg)+SIRP-α construct (200 μg). Doses are administered by intraperitoneal(IP) injection at 0.05 mL/mouse on days 1, 4 and 7. Tumor response tocombination therapy is determined daily by measuring tumor volume. If onday 4, tumor volume of mice treated with combination therapy shows nosignificant improvement, tremelimumab is replaced with ipilimumab.Similarly, if on day 7, tumor volume of mice treated with combinationtherapy show no significant improvement, tremelimumab is replaced withipilimumab. It is expected that while tremelimumab and ipilimumab targetthe same checkpoint protein, they have different therapeutic efficaciesand synergistic effects with the SIRP-α construct due to their differingFc regions. Tremelimumab is an IgG2 antibody that may be more effectiveat fixing complement while ipilimumab is an IgG1 antibody that may beuseful in preventing the elimination of activated T-cells.

Example 16—Method of Treating a Cancer Expressing an Epithelial Marker

SIRP-α polypeptide constructs, such as a high affinity SIRP-α D1 variant(e.g., any variant provided in Tables 2, 5, and 6) fused to an IgG Fcvariant, are administered to treat a cancer expressing an epithelialcell marker. Increased phagocytosis resulting from the blockade of CD47signaling, for example by a SIRP-α D1 construct, may depend on thepresence of macrophages. Therefore, administration of a SIRP-α D1polypeptide construct in combination with an antibody targeting anepithelial marker that is expressed in or on a cancer cell is used totreat the cancer reducing the risk of side effects, e.g., phagocytosisof epithelial cells, due to a low abundance of macrophages at the skinperiphery.

Mouse models of a cancer expressing an epithelial marker, for exampleEGFR or EpCAM, are administered a SIRP-α construct in combination withan antibody that targets the epithelial marker, e.g., an anti-EGFRantibody or an anti-EpCAM antibody. Antibodies targeting epithelialmarkers can recognize both cancerous cells and non-cancerous cells, forexample non-cancerous cells at the skin periphery. However, it isexpected that non-cancerous cells at the skin periphery will not besusceptible to phagocytosis due to a low abundance of macrophages nearthe skin.

Example 17—Phagocytosis by Single Arm SIRP-α-Fc Fusions

To obtain quantitative measurements of phagocytosis induced by SIRP-α-Fcfusions having a single SIRP-α molecule (e.g., a single arm molecule)(depicted in FIGS. 1, 4A, and 4B), a phagocytosis assay with differentcell types MM1R and N87 cells was performed using methods as describedin Example 8.

Six single arm constructs were tested for in vitro phagocytosis. Thesesingle-arm constructs are generated using knob & hole strategies.Homodimer SIRP-α Fc fusion of SEQ ID NO: 136 was used as a double armcomparison (control). A first single-arm SIRP-α Fc fusion (e.g., A) wasformed from a heterodimer of SEQ ID NO: 139 (an Fc variant) and SEQ IDNO: 138 (a SIRP-α Fc fusion). A second single-arm SIRP-α Fc fusion(e.g., B) was formed from a heterodimer of SEQ ID NO: 141 (an Fcvariant) and SEQ ID NO: 140 (a SIRP-α Fc fusion). A third single-armSIRP-α Fc fusion (e.g., C) was formed from a heterodimer of SEQ ID NO:139 (an Fc variant) and SEQ ID NO: 142 (a SIRP-α Fc fusion). A fourthsingle-arm SIRP-α Fc fusion (e.g., D) was formed from a heterodimer ofSEQ ID NO: 141 (an Fc variant) and SEQ ID NO: 143 (a SIRP-α Fc fusion).A fifth single-arm SIRP-α Fc fusion (e.g., E) was formed from aheterodimer of SEQ ID NO: 147 (an Fc variant) and SEQ ID NO: 146 (aSIRP-α Fc fusion). A sixth single-arm SIRP-α Fc fusion (e.g., F) wasformed from a heterodimer of SEQ ID NO: 149 (an Fc variant) and SEQ IDNO: 148 (a SIRP-α Fc fusion). The CD47 binding affinities (K_(D)) of theSIRP-α single-arm when tested as a monomer are as follows: ˜10 pM (A,B),˜100 pM (C, D) and ˜5 nM (E, F). The sequences are provided in Table 27below.

TABLE 27 Amino Acid Sequences of SIRPα - Fc Fusionsfor the Construction of Heterodimers SEQ ID NO: Amino Acid Sequence 138EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 139DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 140EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 141DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 142EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 143EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRELIYNQREGPFPRVTTVSDTTKRNNMDFSIRIGAITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 146EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 147DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 148EEELQVIQPDKSVLVAAGETATLRCTATSLFPVGPIQWFRGAGPGRELIYNQRQGPFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 149DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK

FIGS. 15-17 shows phagocytosis of multiple myeloma line 1R (MM1R) andgastric carcinoma line N87 by non-polarized, human monocyte-derivedmacrophages. “+” and “−” denotes the addition or absence of Daratumumab(Dara) respectively in FIGS. 15-16. In FIG. 17, “+” and “−” denotesaddition and absence of Herceptin/trastuzumab (Her) respectively.

FIG. 15 shows construct A in the presence of a control antibody(IgG1,k), e.g., “A−” has ablated phagocytosis as a single agent while itis able to increase Daratumumab (Dara) phagocytosis, e.g., “A+”.Similarly, construct B in the presence of a control antibody (IgG1,k),e.g., “B−” has ablated phagocytosis as a single agent while it is ableto increase Daratumumab (Dara) phagocytosis, e.g., “B+”. The percent ofmacrophages that phagocytosed MM1R and are CFSE⁺ is indicated on they-axis. Concentration of CD47 binding sites from addition of constructA, B, or control construct is indicated on the x-axis. The levels ofphagocytosis are comparable to the control construct (which is adouble-arm SIRP-α). The level of phagocytosis resulting from incubationwith an anti-CD47 antibody, e.g. B6H12 (100 nM), is comparable toincubation with Dara, e.g. PBS+. As shown, single arm SIRP-α-Fc fusionscan increase Dara phagocytosis comparable to a double arm SIRP-α-Fcfusions.

FIG. 16 shows construct C in the presence of a control antibody(IgG1,k), e.g., “C−” has ablated phagocytosis in the phagocytosis assayas a single agent while it is able to increase Daratumumab (Dara)phagocytosis, e.g., “C+”. Similarly, construct D in the presence of acontrol antibody (IgG1,k), e.g., “D−” has ablated phagocytosis in thephagocytosis assay as a single agent while it is able to increaseDaratumumab (Dara) phagocytosis, e.g., “D+”. The percent of macrophagesthat phagocytosed MM1R and are CFSE⁺ is indicated on the y-axis.Concentration of CD47 binding sites from addition of construct C, D, orcontrol construct is indicated on the x-axis. The levels of phagocytosisare comparable to the control construct (which is a double-arm SIRP-α).The level of phagocytosis resulting from incubation with an anti-CD47antibody, e.g. B6H12 (100 nM), is comparable to incubation with Dara,e.g. PBS+. As shown, single arm SIRP-α-Fc fusions can increase Daraphagocytosis comparable to a double arm SIRP-α-Fc fusions. As shown,single arm SIRP-α-Fc fusions can increase Dara phagocytosis comparableto a double arm SIRP-α-Fc fusions.

FIG. 17 shows phagocytosis in the presence of low affinity single-armSIRP-α constructs (E, F) performed similarly as above examples. Asshown, these low affinity single-arm SIRP-α constructs (E, F) incombination with Herceptin showed comparable phagocytosis of N87 cellsto Herceptin alone (PBS+). Therefore, 5 nM affinity for CD47 is notsufficient for single-arm SIRP-α-Fc fusion to enhance further in vitrophagocytosis in combination with Herceptin.

Example 17—Cross Reactivity of High Affinity SIRP-α D1 Variants

Polypeptides of high affinity SIRP-α D1 variants were generated aspreviously described. Binding to human, mouse, and rat CD47 wasdetermined using SPR as measured by a Biacore T100 instrument (GEHealthcare) and Proteon XPR36 (Bio-rad, Hercules, Calif.) as describedin Example 1. SEQ ID NO: 215 is an engineered SIRP-α D1 variant thatdoes not bind to human, mouse, or rat CD47 and was utilized as anegative control.

TABLE 28 Representative Cross-Species CD47 Binding Affinity for HighAffinity SIRP-α Variants K_(D) (M) SEQ ID NO: Human Mouse Rat 85  2.03 ×10⁻¹⁰ 2.16 × 10⁻⁹ 1.96 × 10⁻⁸ 198  1.55 × 10⁻¹⁰ 1.41 × 10⁻⁹ 9.88 × 10⁻⁹199 1.26 × 10⁻⁹ 1.25 × 10⁻⁹ 1.07 × 10⁻⁸ 200  3.04 × 10⁻¹⁰ 1.17 × 10⁻⁹1.42 × 10⁻⁸ 204  6.53 × 10⁻¹⁰  4.48 × 10⁻¹⁰ 3.42 × 10⁻⁹ 205  2.48 ×10⁻¹⁰  5.69 × 10⁻¹⁰ 4.28 × 10⁻⁹ 206  9.67 × 10⁻¹⁰  2.88 × 10⁻¹⁰ 1.49 ×10⁻⁹ 207 1.04 × 10⁻⁹  8.80 × 10⁻¹⁰ 5.90 × 10⁻⁹ 208  2.19 × 10⁻¹⁰  8.32 ×10⁻¹⁰ 5.17 × 10⁻⁹ 209 1.01 × 10⁻⁹  3.71 × 10⁻¹⁰ 2.26 × 10⁻⁹ 210 1.65 ×10⁻⁹  6.12 × 10⁻¹⁰ 4.59 × 10⁻⁹ 136 1.3904 × 10⁻¹⁰   1.1407 × 10⁻⁸   6.43× 10⁻⁹ 214 2.00 × 10⁻⁹ 6.30 × 10⁻⁸ 8.00 × 10⁻⁸ 215 Non-BindingNon-Binding Non-Binding

SEQ ID NO: 215: EEELQVIQPDKSVLVAAGETATLRCTATSLIPRGPIQWFRGAGPGRELIYNRKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Example 18—Anti-Tumor Activity of High Affinity SIRP-α Constructs in aMouse Xenograft Tumor Model

Immunodeficient NOD scid gamma (NSG) mice(NOD.Cg-Prkdc^(scid)Il2rg^(tm1Wjl)/SzJ; 50 females, plus spares) werepurchased as 6- to 10-week-old animals. Human lymphoma cell lineGFP-Luc-Raji cells were grown in RPMI 1640 containing 10% fetal bovineserum, penicillin, streptomycin, and L-glutamine. Cells then were spundown and re-suspended at a concentration of 1.0×10⁷ cells/mL inserum-free medium without additives and combined 1:1 with Matrigel™(Trevigen, Gaithersburg, Md.). On Day −11 (i.e., 11 days before theprojected staging day), the mice were implanted by subcutaneousinjection into the left flank with 200 μL (1.0×10⁶ cells) per mouse ofthe freshly prepared GFP-Luc-Raji:Matrigel mixture. When the tumorsreached a mean volume of approximately 55 mm³, fifty animals withestablished tumors and moderate body weights were randomized into 5treatment groups (Group 1-5, n=10 mice each). Starting on Day 1, Groups1 to 5 were treated with (1) SEQ ID NO: 215 [10 mg/kg (mpk), 3×/week];(2) SEQ ID NO: 104 (10 mpk, 3×/week); (3) rituximab (5 mpk, 2×/week)+SEQID NO: 100 (10 mpk, 3×/week); (4) rituximab (5 mpk, 2×/week)+SEQ ID NO:104 (10 mpk, 3×/week); or (5) rituximab (5 mpk, 2×/week)+SEQ ID NO: 215(10 mpk, 3×/week), respectively. Doses were administered byintraperitoneal (IP) injection at 0.05 mL/mouse. For all animals, doseswere administered starting on the staging day and continuing for a totalof 31 days (Days 1-31).

Clinical observations were recorded twice per day (morning and evening).Additional findings were recorded as observed. Body weights weremeasured three times per week using an electronic balance (Ohaus SCOUT®PRO). Tumor sizes were measured three times per week, and at studycompletion, using microcalipers (Mitutoyo, Aurora, Ill.) to measure theperpendicular minor (width, W, and height, H) and major (length, L)dimensions. Tumor volume (mm³) was calculated using the formula for thevolume of an ellipsoid sphere (L×W×H/2). Blood samples were drawn from20 animals on Day 1 (baseline; prior to group assignment) and from allanimals on Day 8 (Week 1) and Day 31 (at termination). Blood specimenswere submitted for complete blood counts (CBCs) on the respective day ofdraw.

The SIRP-α construct of SEQ ID NO: 215 does not exhibit measurablebinding to CD47 (see Table 28). Tumors in the SEQ ID NO: 215-dosed group(Group 1) grew linearly through Day 31 (FIG. 19A), similar to tumorsobserved in the PBS vehicle group of the same model (data not shown).This observation demonstrates ongoing tumor growth in the absence ofeffective treatment.

Comparisons between Groups 1 and 5 (SEQ ID NO: 215 with or withoutrituximab) and between Groups 2 and 4 (SEQ ID NO: 104 with or withoutrituximab) reveal that the combination treatments yielded significantattenuation of tumor volume, both as raw values (from Day 9) andnormalized values (from Day 7). By Day 16, the majority of mice in Group3 (SEQ ID NO: 100+rituximab) and Group 4 (SEQ ID NO: 104+rituximab) nolonger harbored detectable tumors; these two combination treatmentsshowed similar efficacy. In contrast, tumor growth appeared to recoverin animals of Group 5 (SEQ ID NO: 215+rituximab) from Day 18 on. Tumorvolumes of all five groups over the study period (mean+/−SEM andindividual scatter plots) are presented in FIG. 19A and FIG. 19Brespectively.

Complete blood count (CBC) values (red blood cells, hemoglobin,hematocrit, platelets, etc.) measured pre-dose (Day 1), 1 week afterdosing (Day 9), and 4 weeks after dosing (Day 31). Parameters did notdiffer significantly at Week 1 or Week 4 among the five groups.Hemoglobin (HGB) values are shown in FIG. 19C. These results demonstratethat high affinity SIRP-α constructs can effectively attenuate tumorgrowth and synergize with rituximab in an in vivo mouse model of cancer.Furthermore, in contrast to anti-CD47 based antibody treatments, noacute episodes of anemia were observed in any of the test groups treatedwith the high affinity SIRP-α constructs.

Example 19: SIRP-α Fc Variant Constructs Exhibit Decreased Red BloodCell Toxicity

Red blood cell loss is a concern when targeting CD47. To examine theeffects of a SIRP-α Fc variant construct on red blood cell toxicity,mice were treated with a high affinity SIRP-α variant constructcontaining either a wildtype IgG1 Fc construct (SEQ ID NO: 216) or aIgG1 Fc variant construct (SEQ ID NO:96) with IgG1 mutations L234A,L235A, G237A, and N297A (IgG1_AAA_N297A). Mice were assigned to fivegroups of six and were treated on day 1 and 7 (see solid arrows in FIG.20) with either: (1) PBS; (2) 10 mg/kg SEQ ID NO: 216 (wildtype IgG1Fc); (3) 30 mg/kg SEQ ID NO: 216; (4) 10 mg/kg SEQ ID NO: 96(IgG1_AAA_N297A); or (5) 30 mg/kg SEQ ID NO: 96. Baseline complete bloodcount (CBC) measurements were taken from all animals on day −7 and forthree of six animals on day 1. The blood draws (see FIG. 20) rotatedbetween three mice from each group to not exceed the amount of bloodwithdrawal allowed per week. As demonstrated in FIG. 20, treatment witha wildtype IgG1 containing SIRP-α D1 variant construct resulted in adose-dependent decrease in red blood cell counts. Conversely, treatmentwith an IgG1_AAA_N297A containing SIRP-α D1 variant construct resultedin red blood cell counts similar to the PBS treated control group.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein can be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1-62. (canceled) 63: A polypeptide, comprising: (a) a CD47 bindingpolypeptide; and (b) an Fc variant, wherein the Fc variant is selectedfrom: (i) a human IgG1 Fc region comprising mutations L234A, L235A,G237A, and N297A; (ii) a human IgG2 Fc region comprising mutationsA330S, P331S and N297A; or (iii) a human IgG4 Fc region comprisingmutations S228P, E233P, F234V, L235A, delG236, and N297A, whereinnumbering is according to the EU index.
 64. (canceled) 65: Thepolypeptide of claim 63, wherein the Fc variant is a human IgG1 Fcregion comprising mutations L234A, L235A, G237A, and N297A. 66: Thepolypeptide of claim 63, wherein the Fc variant is a human IgG2 Fcregion comprising mutations A330S, P331S and N297A. 67-69. (canceled)70: The polypeptide of claim 63, wherein the Fc variant is a human IgG4Fc region comprising mutations S228P, E233P, F234V, L235A, delG236, andN297A. 71-72. (canceled) 73: The polypeptide of claim 63, wherein the Fcvariant binds to an Fcγ receptor with a K_(D) greater than about 5×10⁻⁶M. 74-75. (canceled) 76: The polypeptide of claim 63, wherein thepolypeptide does not cause acute anemia in rodents and non-humanprimates. 77: The polypeptide of claim 63, wherein the polypeptide doesnot cause acute anemia in humans. 78: The polypeptide of claim 63,wherein the CD47 binding polypeptide is a signal-regulatory protein α(SIRP-α) polypeptide or a fragment thereof that is capable of bindingCD47. 79: The polypeptide of claim 63, wherein the SIRP-α polypeptidecomprises a SIRP-α D1 variant comprising the amino acid sequence,EEELQX₁IQPDKSVLVAAGETATLRCTX₂TSLX₃PVGPIQWFRGAGPGRX₄LIYNQXsEGX₆FPRVTTVSDX₇TKRNNMDFSIRIGXsITPADAGTYYCX₉KFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ IDNO: 51), wherein X₁ is V or I; X₂ is A or I; X₃ is I or F; X₄ is E or V;X₅ is K or R; X₆ is H or P; X₇ is L or T; X₈ is any amino acid otherthan N; and X₉ is V or I. 80: The polypeptide of claim 79, wherein theSIRP-α polypeptide comprises a SIRP-α D1 variant wherein X₁ is V or I;X₂ is A or I; X₃ is I or F; X₄ is E; X₅ is K or R; X₆ is H or P; X₇ is Lor T; X₈ is not N; and X₉ is V. 81-93. (canceled) 94: A method oftreating an individual having a disease or disorder, the methodcomprising administering to the individual a polypeptide comprising: (a)a CD47 binding polypeptide; and (b) an Fc variant, wherein the Fcvariant is selected from the group consisting of: (i) a human IgG1 Fcregion comprising mutations L234A, L235A, G237A, and N297A; (ii) a humanIgG2 Fc region comprising mutations A330S, P331S and N297A; (iii) ahuman IgG4 Fc region comprising mutations S228P, E233P, F234V, L235A,delG236, and N297A, wherein numbering is according to the EU index. 95:The method of claim 94, wherein the disease or disorder is a cancer, anautoimmune disease, or an inflammatory disease. 96: The method of claim94, wherein the disease or disorder is a cancer, and the cancer isselected from solid tumor cancer, hematological cancer, acute myeloidleukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acutelymphoblastic leukemia, non-Hodgkin lymphoma, Hodgkin lymphoma, multiplemyeloma, bladder cancer, pancreatic cancer, cervical cancer, endometrialcancer, lung cancer, bronchus cancer, liver cancer, ovarian cancer,colon and rectal cancer, stomach cancer, gastric cancer, gallbladdercancer, gastrointestinal stromal tumor cancer, thyroid cancer, head andneck cancer, oropharyngeal cancer, esophageal cancer, melanoma,non-melanoma skin cancer, Merkel cell carcinoma, virally induced cancer,neuroblastoma, breast cancer, prostate cancer, renal cancer, renal cellcancer, renal pelvis cancer, leukemia, lymphoma, sarcoma, glioma, braintumor, and carcinoma. 97: The method of claim 94, wherein the disease ordisorder is an autoimmune disease or an inflammatory disease, and theautoimmune disease or the inflammatory disease is selected from multiplesclerosis, rheumatoid arthritis, a spondyloarthropathy, systemic lupuserythematosus, an antibody-mediated inflammatory or autoimmune disease,graft versus host disease, sepsis, diabetes, psoriasis, atherosclerosis,Sjogren's syndrome, progressive systemic sclerosis, scleroderma, acutecoronary syndrome, ischemic reperfusion, Crohn's Disease, endometriosis,glomerulonephritis, myasthenia gravis, idiopathic pulmonary fibrosis,asthma, acute respiratory distress syndrome (ARDS), vasculitis, andinflammatory autoimmune myositis. 98: The method of claim 94, whereinthe CD47 binding polypeptide comprises a sequence set forth in any oneof SEQ ID NOs: 78-85. 99: The method of claim 98, further comprisingadministration of at least one additional agent. 100: The method ofclaim 99, wherein the at least one additional agent is an antibody,tumor associated antigen, or a non-antibody therapeutic. 101: The methodof claim 100, wherein at least two additional agents are administered.102: The method of claim 101, wherein the at least two additional agentscomprise two antibodies. 103: The method of claim 101, wherein the atleast two additional agents comprise an antibody and a tumor associatedantigen.
 104. (canceled) 105: The method of claim 100, wherein theantibody is a human IgG1 isotype antibody, a human IgG2 isotypeantibody, or a human IgG4 isotype antibody. 106-107. (canceled) 108: Themethod of claim 100, wherein the antibody is selected from an anti-HER2antibody, anti-CD20 antibody, anti-CD19 antibody, anti-CS1 antibody,anti-CD38 antibody, anti-EGFR antibody, anti-OX40 antibody, anti-PD-1antibody, anti-PD-L1 antibody, anti-RANKL antibody, anti-CD274 antibody,anti-CTLA-4 antibody, anti-CD137 antibody, anti-4-1BB antibody,anti-B7-H3 antibody, anti-FZD7 antibody, anti-CD27 antibody, anti-CCR4antibody, anti-CSF1R antibody, anti-CSF antibody, anti-CD30 antibody,anti-BAFF antibody, anti-VEGF antibody, or anti-VEGFR2 antibody.109-146. (canceled) 147: The method of claim 99, wherein the at leastone additional agent is a tumor associated antigen and the tumorassociated antigen elicits an immune response. 148: The method of claim99, wherein the at least one additional agent is an antibody and theantibody targets a HLA/peptide or MHC/peptide complex. 149: The methodof claim 148, wherein the antibody targets a HLA/peptide or MHC/peptidecomplex comprising NY-ESO-1/LAGE1, SSX-2, MAGE family (MAGE-A3),gp100/pmeli7, Melan-A/MART-1, gp75/TRP1, tyrosinase, TRP2, CEA, PSA,TAG-72, Immature laminin receptor, MOK/RAGE-1, WT-1, Her2/neu, EphA3,SAP-1, BING-4, Ep-CAM, MUC1, PRAME, survivin, Mesothelin, BRCA1/2, CDK4,CML66, MART-2, p53, Ras, β-catenin, TGF-βRII, HPV E6, or E7. 150: Themethod of claim 148, wherein the antibody is ESK1, RL1B, Pr20, or 3.2G1.151-156. (canceled) 157: A nucleic acid encoding a polypeptidecomprising (a) a CD47 binding polypeptide; and (b) an Fc variant,wherein the Fc variant is selected from the group consisting of: (i) ahuman IgG1 Fc region comprising mutations L234A, L235A, G237A, andN297A; (ii) a human IgG2 Fc region comprising mutations A330S, P331S andN297A (iii) a human IgG4 Fc region comprising mutations S228P, E233P,F234V, L235A, delG236, and N297A, wherein numbering is according to theEU index. 158: A vector comprising the nucleic acid of claim
 157. 159: Ahost cell comprising the nucleic acid of claim
 157. 160: A method ofproducing a polypeptide comprising culturing the host cell of claim 159under appropriate conditions to cause expression of the polypeptide andrecovering the polypeptide. 161: A pharmaceutical compositioncomprising: (I) a polypeptide that comprises (a) a CD47 bindingpolypeptide; and (b) an Fc variant, wherein the Fc variant is selectedfrom the group consisting of: (i) a human IgG1 Fc region comprisingmutations L234A, L235A, G237A, and N297A; (ii) a human IgG2 Fc regioncomprising mutations A330S, P331S and N297A (iii) a human IgG4 Fc regioncomprising mutations S228P, E233P, F234V, L235A, delG236, and N297A,wherein numbering is according to the EU index; and (II) apharmaceutically acceptable carrier. 162: A dimer comprising apolypeptide that comprises: (a) a CD47 binding polypeptide; and (b) anFc variant, wherein the Fc variant is selected from the group consistingof: (i) a human IgG1 Fc region comprising mutations L234A, L235A, G237A,and N297A; (ii) a human IgG2 Fc region comprising mutations A330S, P331Sand N297A (iii) a human IgG4 Fc region comprising mutations S228P,E233P, F234V, L235A, delG236, and N297A, wherein numbering is accordingto the EU index. 163: The dimer of claim 162, wherein the dimer is aheterodimer. 164: The dimer of claim 162, wherein the dimer is ahomodimer.